Covalently Modified Surfaces, Kits, and Methods of Preparation and Use

ABSTRACT

In biosciences and related fields, it can be useful to modify surfaces of apparatuses, devices, and materials that contact biomaterials such as biomolecules and biological micro-objects. Described herein are surface modifying and surface functionalizing reagents, preparation thereof, and methods for modifying surfaces to provide improved or altered performance with biomaterials.

This application is a continuation of International Patent ApplicationNo. PCT/US2017/034832, filed May 26, 2017, which claims the benefitunder 35 U.S.C. 119(e) of U.S. Provisional Application No. 62/342,131,filed on May 26, 2016; U.S. Provisional Application No. 62/345,603,filed on Jun. 3, 2016; 62/353,938, filed on Jun. 23, 2016; U.S.Provisional Application No. 62/411,191, filed on Oct. 21, 2016; and ofU.S. Provisional Application No. 62/410,238, filed on Oct. 19, 2016,each of which disclosures is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

In biosciences and related fields, it can be useful to modify surfacesof apparatuses, devices, and materials that contact biomaterials such asbiomolecules and biological micro-objects. Some embodiments of thepresent invention include a siloxane reagent, preparation thereof, andmethods for modifying surfaces to provide improved or alteredperformance with biomaterials.

SUMMARY OF THE INVENTION

In a first aspect, a microfluidic device is provided, where themicrofluidic device includes an enclosure comprising a base, a cover,and microfluidic circuit material defining a fluidic circuit therein,where at least one inner surface of the base, the cover and themicrofluidic circuit material has a first covalently bound surfacemodification including a first linking group, and a first moiety,wherein the first moiety is a first surface contact moiety or a firstreactive moiety; where at least one inner surface of the base, the coverand the microfluidic circuit material has a second covalently boundsurface modification including a second linking group, and a secondmoiety, where the second moiety is a second surface contact moiety orsecond reactive moiety, and where the first linking group and the secondlinking group are different from each other and/or the first moiety isdifferent from the second moiety. In some embodiments, a common innersurface of the base, the cover and the microfluidic circuit material hasthe first covalently bound surface modification and the secondcovalently bound surface modification.

In another aspect, a method of forming a covalently modified surface onat least one inner surface of a microfluidic device including anenclosure having a base, a cover and microfluidic circuit materialdefining a fluidic circuit therein, the method including: contacting theat least one inner surface with a first modifying reagent and a secondmodifying reagent; reacting the first modifying reagent with a firstnucleophilic moiety of the at least one inner surface; reacting thesecond modifying reagent with a second nucleophilic moiety of the atleast one inner surface; and forming the at least one covalentlymodified surface including a first covalently bound surface modificationincluding a first linking group and a first moiety that is a firstsurface contact moiety or a first reactive moiety; and a secondcovalently bound surface modification including a second linking groupand a second moiety that is a second surface contact moiety or secondreactive moiety, where the first linking group is different from thesecond linking group or the first moiety is different from the secondmoiety. In some embodiments, the first covalently bound surfacemodification and the second covalently bound surface modification may beformed on a common inner surface of the base, the cover and themicrofluidic circuit material.

In another aspect, a method is provided for forming different covalentlymodified surfaces in a regioselective manner within a microfluidicdevice. The microfluidic device can include an enclosure having a base,a cover, and a microfluidic circuit material defining a microfluidiccircuit therein, where the microfluidic circuit comprises a flow regionand a sequestration pen, and where the sequestration pen comprises anisolation region and a connection region, the connection regioncomprising a proximal opening to the flow region and fluidicallyconnecting the isolation region to the flow region. The method caninclude the steps of: flowing a first modifying reagent through the flowregion under conditions such that the first modifying reagent does notenter the isolation region of the sequestration pen; reacting the firstmodifying reagent with nucleophilic moieties on at least one surface ofthe flow region, thereby forming a first modified surface within theflow region, wherein the first modified surface does not extend into theisolation region of the sequestration pen; flowing a second modifyingreagent through the flow region under conditions such that the secondmodifying reagent enters into the isolation region of the sequestrationpen; and reacting the second modifying reagent with nucleophilicmoieties on at least one surface of the isolation region of thesequestration pen, thereby forming a second modified surface within theisolation region of the sequestration pen. Typically, the firstmodifying reagent does not have the same structure as the secondmodifying reagent.

In another aspect, a kit is provided, including a microfluidic device asdescribed herein. The kit may further include a surface modifyingreagent having a structure of Formula XII:

RP-L-surface contact moiety  Formula XII;

wherein RP is a reaction pair moiety; surface contact moiety is a moietyconfigured to support cell growth, viability, portability, or anycombination thereof; L is a linker; wherein L may be a bond or 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms, and may further include 0or 1 coupling groups CG.

In another aspect, a compound having a structure of Formula XIII isprovided:

where h is an integer of 1 to 19 and R is selected independently fromthe group consisting of H and C₁-C₆ alkyl. In some embodiments, h is 5to 19.

In yet another aspect, a method of synthesizing a compound having astructure of Formula XIII is provided:

including reacting a compound having a structure of the followingformula:

with a compound having a structure of the formula HSi(OR)₃, in thepresence of a catalyst or an initiator, thereby producing the compoundof Formula XIII, where h is an integer of 1 to 19 and each instance of Ris independently H or C₁ to C₆ alkyl.

In a further aspect, a compound having a structure of Formula IV isprovided:

where n is an integer of 3 to 21, and R is independently H or C₁ to C₆alkyl. In some embodiments, n is 9, 14 or 16.

In another aspect, a method of synthesizing a compound of Formula IV isprovided:

including the step of reacting a compound having a structure of FormulaXIII:

where h is 1 to 19 with azide ion, thereby producing the compound ofFormula IV, where n is 3 to 21 and R is H or C₁-C₆ alkyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an example of a system for use with a microfluidicdevice and associated control equipment according to some embodiments ofthe disclosure.

FIGS. 1B and 1C illustrate a microfluidic device according to someembodiments of the disclosure.

FIGS. 2A and 2B illustrate isolation pens according to some embodimentsof the disclosure.

FIG. 2C illustrates a detailed sequestration pen according to someembodiments of the disclosure.

FIGS. 2D-F illustrate sequestration pens according to some otherembodiments of the disclosure.

FIG. 2G illustrates a microfluidic device according to an embodiment ofthe disclosure.

FIG. 2H illustrates a coated surface of the microfluidic deviceaccording to an embodiment of the disclosure.

FIG. 3A illustrates a specific example of a system for use with amicrofluidic device and associated control equipment according to someembodiments of the disclosure.

FIG. 3B illustrates an imaging device according to some embodiments ofthe disclosure.

FIG. 4 is a graphical representation of a FTIR spectrum for modifiedmicrofluidic circuit material according to some embodiments of thedisclosure.

FIGS. 5A and 5B are graphical representation of overlaid FTIR formodified surfaces according to some embodiments of the disclosure.

FIGS. 6A to 6B are photographic representations of cell culturing andcell unpenning according to an embodiment of the invention.

FIGS. 7A to 7B are photographic representations of cell culturing andcell unpenning according to another embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This specification describes exemplary embodiments and applications ofthe disclosure. The disclosure, however, is not limited to theseexemplary embodiments and applications or to the manner in which theexemplary embodiments and applications operate or are described herein.Moreover, the figures may show simplified or partial views, and thedimensions of elements in the figures may be exaggerated or otherwisenot in proportion. In addition, as the terms “on,” “attached to,”“connected to,” “coupled to,” or similar words are used herein, oneelement (e.g., a material, a layer, a substrate, etc.) can be “on,”“attached to,” “connected to,” or “coupled to” another elementregardless of whether the one element is directly on, attached to,connected to, or coupled to the other element or there are one or moreintervening elements between the one element and the other element.Also, unless the context dictates otherwise, directions (e.g., above,below, top, bottom, side, up, down, under, over, upper, lower,horizontal, vertical, “x,” “y,” “z,” etc.), if provided, are relativeand provided solely by way of example and for ease of illustration anddiscussion and not by way of limitation. In addition, where reference ismade to a list of elements (e.g., elements a, b, c), such reference isintended to include any one of the listed elements by itself, anycombination of less than all of the listed elements, and/or acombination of all of the listed elements. Section divisions in thespecification are for ease of review only and do not limit anycombination of elements discussed.

Where dimensions of microfluidic features are described as having awidth or an area, the dimension typically is described relative to anx-axial and/or y-axial dimension, both of which lie within a plane thatis parallel to the substrate and/or cover of the microfluidic device.The height of a microfluidic feature may be described relative to az-axial direction, which is perpendicular to a plane that is parallel tothe substrate and/or cover of the microfluidic device. In someinstances, a cross sectional area of a microfluidic feature, such as achannel or a passageway, may be in reference to a x-axial/z-axial, ay-axial/z-axial, or an x-axial/y-axial area.

As used herein, “substantially” means sufficient to work for theintended purpose. The term “substantially” thus allows for minor,insignificant variations from an absolute or perfect state, dimension,measurement, result, or the like such as would be expected by a personof ordinary skill in the field but that do not appreciably affectoverall performance. When used with respect to numerical values orparameters or characteristics that can be expressed as numerical values,“substantially” means within ten percent.

The term “ones” means more than one. As used herein, the term“plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain radical consisting solely of carbon and hydrogen atoms, containingno unsaturation, having from one to six carbon atoms (e.g., C₁-C₆alkyl). Whenever it appears herein, a numerical range such as “1 to 6”refers to each integer in the given range; e.g., “1 to 6 carbon atoms”means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms,3 carbon atoms, etc., up to and including 6 carbon atoms, although thepresent definition also covers the occurrence of the term “alkyl” whereno numerical range is designated. In some embodiments, it is a C₁-C₃alkyl group. Typical alkyl groups include, but are in no way limited to,methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butylisobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl, and thelike. The alkyl is attached to the rest of the molecule by a singlebond, for example, methyl (Me), ethyl (Et), n-propyl, 1-methylethyl(iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), hexyl, andthe like.

Unless stated otherwise specifically in the specification, an alkylgroup may be optionally substituted by one or more substituents whichindependently are: aryl, arylalkyl, heteroaryl, heteroarylalkyl,hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro,trimethylsilanyl, —OR′, —SR′, —OC(O)—R′, —N(R′)₂, —C(O)R′, —C(O)OR′,—OC(O)N(R′)₂, —C(O)N(R′)₂, —N(R′)C(O)OR′, —N(R′)C(O)R′,—N(R′)C(O)N(R′)₂, N(R′)C(NR′)N(R′)₂, —N(R′)S(O)_(t)R′(where t is 1 or2), —S(O)_(t)OR′ (where t is 1 or 2), —S(O)_(t)N(R′)₂ (where t is 1 or2), or PO₃(R′)₂ where each R′ is independently hydrogen, alkyl,fluoroalkyl, aryl, aralkyl, heterocycloalkyl, or heteroaryl.

As referred to herein, a fluorinated alkyl moiety is an alkyl moietyhaving one or more hydrogens of the alkyl moiety replaced by a fluorosubstituent. A perfluorinated alkyl moiety has all hydrogens attached tothe alkyl moiety replaced by fluoro substituents.

As referred to herein, a “halo” moiety is a bromo, chloro, or fluoromoiety.

As referred to herein, an “olefinic” compound is an organic moleculewhich contains an “alkene” moiety. An alkene moiety refers to a groupconsisting of at least two carbon atoms and at least one carbon-carbondouble bond. The non-alkene portion of the molecule may be any class oforganic molecule, and in some embodiments, may include alkyl orfluorinated (including but not limited to perfluorinated) alkylmoieties, any of which may be further substituted.

As used herein, “air” refers to the composition of gases predominatingin the atmosphere of the earth. The four most plentiful gases arenitrogen (typically present at a concentration of about 78% by volume,e.g., in a range from about 70-80%), oxygen (typically present at about20.95% by volume at sea level, e.g. in a range from about 10% to about25%), argon (typically present at about 1.0% by volume, e.g. in a rangefrom about 0.1% to about 3%), and carbon dioxide (typically present atabout 0.04%, e.g., in a range from about 0.01% to about 0.07%). Air mayhave other trace gases such as methane, nitrous oxide or ozone, tracepollutants and organic materials such as pollen, diesel particulates andthe like. Air may include water vapor (typically present at about 0.25%,or may be present in a range from about 10 ppm to about 5% by volume).Air may be provided for use in culturing experiments as a filtered,controlled composition and may be conditioned as described herein.

As used herein, the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10,or more.

As used herein, the term “disposed” encompasses within its meaning“located.”

As used herein, a “microfluidic device” or “microfluidic apparatus” is adevice that includes one or more discrete microfluidic circuitsconfigured to hold a fluid, each microfluidic circuit comprised offluidically interconnected circuit elements, including but not limitedto region(s), flow path(s), channel(s), chamber(s), and/or pen(s), andat least one port configured to allow the fluid (and, optionally,micro-objects suspended in the fluid) to flow into and/or out of themicrofluidic device. Typically, a microfluidic circuit of a microfluidicdevice will include a flow region, which may include a microfluidicchannel, and at least one chamber, and will hold a volume of fluid ofless than about 1 mL, e.g., less than about 750, 500, 250, 200, 150,100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μL. In certainembodiments, the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5,2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75,10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 μL. Themicrofluidic circuit may be configured to have a first end fluidicallyconnected with a first port (e.g., an inlet) in the microfluidic deviceand a second end fluidically connected with a second port (e.g., anoutlet) in the microfluidic device.

As used herein, a “nanofluidic device” or “nanofluidic apparatus” is atype of microfluidic device having a microfluidic circuit that containsat least one circuit element configured to hold a volume of fluid ofless than about 1 μL, e.g., less than about 750, 500, 250, 200, 150,100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less. Ananofluidic device may comprise a plurality of circuit elements (e.g.,at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). Incertain embodiments, one or more (e.g., all) of the at least one circuitelements is configured to hold a volume of fluid of about 100 pL to 1nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g.,all) of the at least one circuit elements are configured to hold avolume of fluid of about 20 nL to 200 nL, 100 to 200 nL, 100 to 300 nL,100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to600 nL, or 250 to 750 nL.

A microfluidic device or a nanofluidic device may be referred to hereinas a “microfluidic chip” or a “chip”; or “nanofluidic chip” or “chip”.

A “microfluidic channel” or “flow channel” as used herein refers to flowregion of a microfluidic device having a length that is significantlylonger than both the horizontal and vertical dimensions. For example,the flow channel can be at least 5 times the length of either thehorizontal or vertical dimension, e.g., at least 10 times the length, atleast 25 times the length, at least 100 times the length, at least 200times the length, at least 500 times the length, at least 1,000 timesthe length, at least 5,000 times the length, or longer. In someembodiments, the length of a flow channel is about 100,000 microns toabout 500,000 microns, including any value therebetween. In someembodiments, the horizontal dimension is about 100 microns to about 1000microns (e.g., about 150 to about 500 microns) and the verticaldimension is about 25 microns to about 200 microns, (e.g., from about 40to about 150 microns). It is noted that a flow channel may have avariety of different spatial configurations in a microfluidic device,and thus is not restricted to a perfectly linear element. For example, aflow channel may be, or include one or more sections having, thefollowing configurations: curve, bend, spiral, incline, decline, fork(e.g., multiple different flow paths), and any combination thereof. Inaddition, a flow channel may have different cross-sectional areas alongits path, widening and constricting to provide a desired fluid flowtherein. The flow channel may include valves, and the valves may be ofany type known in the art of microfluidics. Examples of microfluidicchannels that include valves are disclosed in U.S. Pat. Nos. 6,408,878and 9,227,200, each of which is herein incorporated by reference in itsentirety.

As used herein, the term “obstruction” refers generally to a bump orsimilar type of structure that is sufficiently large so as to partially(but not completely) impede movement of target micro-objects between twodifferent regions or circuit elements in a microfluidic device. The twodifferent regions/circuit elements can be, for example, a microfluidicsequestration pen and a microfluidic channel, or a connection region andan isolation region of a microfluidic sequestration pen.

As used herein, the term “constriction” refers generally to a narrowingof a width of a circuit element (or an interface between two circuitelements) in a microfluidic device. The constriction can be located, forexample, at the interface between a microfluidic sequestration pen and amicrofluidic channel, or at the interface between an isolation regionand a connection region of a microfluidic sequestration pen.

As used herein, the term “transparent” refers to a material which allowsvisible light to pass through without substantially altering the lightas is passes through.

As used herein, the term “micro-object” refers generally to anymicroscopic object that may be isolated and/or manipulated in accordancewith the present disclosure. Non-limiting examples of micro-objectsinclude: inanimate micro-objects such as microparticles; microbeads(e.g., polystyrene beads, Luminex™ beads, or the like); magnetic beads;microrods; microwires; quantum dots, and the like; biologicalmicro-objects such as cells; biological organelles; vesicles, orcomplexes; synthetic vesicles; liposomes (e.g., synthetic or derivedfrom membrane preparations); lipid nanorafts, and the like; or acombination of inanimate micro-objects and biological micro-objects(e.g., microbeads attached to cells, liposome-coated micro-beads,liposome-coated magnetic beads, or the like). Beads may includemoieties/molecules covalently or non-covalently attached, such asfluorescent labels, proteins, carbohydrates, antigens, small moleculesignaling moieties, or other chemical/biological species capable of usein an assay. Lipid nanorafts have been described, for example, inRitchie et al. (2009) “Reconstitution of Membrane Proteins inPhospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231.

As used herein, the term “cell” is used interchangeably with the term“biological cell.” Non-limiting examples of biological cells includeeukaryotic cells, plant cells, animal cells, such as mammalian cells,reptilian cells, avian cells, fish cells, or the like, prokaryoticcells, bacterial cells, fungal cells, protozoan cells, or the like,cells dissociated from a tissue, such as muscle, cartilage, fat, skin,liver, lung, neural tissue, and the like, immunological cells, such as Tcells, B cells, natural killer cells, macrophages, and the like, embryos(e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells,cells from a cell line, cancer cells, infected cells, transfected and/ortransformed cells, reporter cells, and the like. A mammalian cell canbe, for example, from a human, a mouse, a rat, a horse, a goat, a sheep,a cow, a primate, or the like.

A colony of biological cells is “clonal” if all of the living cells inthe colony that are capable of reproducing are daughter cells derivedfrom a single parent cell. In certain embodiments, all the daughtercells in a clonal colony are derived from the single parent cell by nomore than 10 divisions. In other embodiments, all the daughter cells ina clonal colony are derived from the single parent cell by no more than14 divisions. In other embodiments, all the daughter cells in a clonalcolony are derived from the single parent cell by no more than 17divisions. In other embodiments, all the daughter cells in a clonalcolony are derived from the single parent cell by no more than 20divisions. The term “clonal cells” refers to cells of the same clonalcolony.

As used herein, a “colony” of biological cells refers to 2 or more cells(e.g. about 2 to about 20, about 4 to about 40, about 6 to about 60,about 8 to about 80, about 10 to about 100, about 20 to about 200, about40 to about 400, about 60 to about 600, about 80 to about 800, about 100to about 1000, or greater than 1000 cells).

As used herein, the term “maintaining (a) cell(s)” refers to providingan environment comprising both fluidic and gaseous components and,optionally a surface, that provides the conditions necessary to keep thecells viable and/or expanding.

As used herein, the term “expanding” when referring to cells, refers toincreasing in cell number.

As referred to herein, “gas permeable” means that the material orstructure is permeable to at least one of oxygen, carbon dioxide, ornitrogen. In some embodiments, the gas permeable material or structureis permeable to more than one of oxygen, carbon dioxide and nitrogen andmay further be permeable to all three of these gases.

A “component” of a fluidic medium is any chemical or biochemicalmolecule present in the medium, including solvent molecules, ions, smallmolecules, antibiotics, nucleotides and nucleosides, nucleic acids,amino acids, peptides, proteins, sugars, carbohydrates, lipids, fattyacids, cholesterol, metabolites, or the like.

As used herein in reference to a fluidic medium, “diffuse” and“diffusion” refer to thermodynamic movement of a component of thefluidic medium down a concentration gradient.

The phrase “flow of a medium” means bulk movement of a fluidic mediumprimarily due to any mechanism other than diffusion. For example, flowof a medium can involve movement of the fluidic medium from one point toanother point due to a pressure differential between the points. Suchflow can include a continuous, pulsed, periodic, random, intermittent,or reciprocating flow of the liquid, or any combination thereof. Whenone fluidic medium flows into another fluidic medium, turbulence andmixing of the media can result.

The phrase “substantially no flow” refers to a rate of flow of a fluidicmedium that, when averaged over time, is less than the rate of diffusionof components of a material (e.g., an analyte of interest) into orwithin the fluidic medium. The rate of diffusion of components of such amaterial can depend on, for example, temperature, the size of thecomponents, and the strength of interactions between the components andthe fluidic medium.

As used herein in reference to different regions within a microfluidicdevice, the phrase “fluidically connected” means that, when thedifferent regions are substantially filled with fluid, such as fluidicmedia, the fluid in each of the regions is connected so as to form asingle body of fluid. This does not mean that the fluids (or fluidicmedia) in the different regions are necessarily identical incomposition. Rather, the fluids in different fluidically connectedregions of a microfluidic device can have different compositions (e.g.,different concentrations of solutes, such as proteins, carbohydrates,ions, or other molecules) which are in flux as solutes move down theirrespective concentration gradients and/or fluids flow through thedevice.

As used herein, a “flow path” refers to one or more fluidicallyconnected circuit elements (e.g. channel(s), region(s), chamber(s) andthe like) that define, and are subject to, the trajectory of a flow ofmedium. A flow path is thus an example of a swept region of amicrofluidic device. Other circuit elements (e.g., unswept regions) maybe fluidically connected with the circuit elements that comprise theflow path without being subject to the flow of medium in the flow path.

As used herein, “isolating a micro-object” confines a micro-object to adefined area within the microfluidic device.

A microfluidic (or nanofluidic) device can comprise “swept” regions and“unswept” regions. As used herein, a “swept” region is comprised of oneor more fluidically interconnected circuit elements of a microfluidiccircuit, each of which experiences a flow of medium when fluid isflowing through the microfluidic circuit. The circuit elements of aswept region can include, for example, regions, channels, and all orparts of chambers. As used herein, an “unswept” region is comprised ofone or more fluidically interconnected circuit element of a microfluidiccircuit, each of which experiences substantially no flux of fluid whenfluid is flowing through the microfluidic circuit. An unswept region canbe fluidically connected to a swept region, provided the fluidicconnections are structured to enable diffusion but substantially no flowof media between the swept region and the unswept region. Themicrofluidic device can thus be structured to substantially isolate anunswept region from a flow of medium in a swept region, while enablingsubstantially only diffusive fluidic communication between the sweptregion and the unswept region. For example, a flow channel of amicro-fluidic device is an example of a swept region while an isolationregion (described in further detail below) of a microfluidic device isan example of an unswept region.

As used herein, a “non-sweeping” rate of fluidic medium flow means arate of flow sufficient to permit components of a second fluidic mediumin an isolation region of the sequestration pen to diffuse into thefirst fluidic medium in the flow region and/or components of the firstfluidic medium to diffuse into the second fluidic medium in theisolation region; and further wherein the first medium does notsubstantially flow into the isolation region.

Surface Modification.

Surfaces of materials, devices, and/or apparatuses for manipulation andstorage of biomaterials may have native properties that are notoptimized for short and/or long term contact with such material, whichmay include but is not limited to micro-objects (including but notlimited to biological micro-objects such as biological cells),biomolecules, fragments of the biomolecules or biological micro-objects,and any combination thereof. It may be useful to modify one or moresurfaces of a material, device or apparatus to decrease one or moreundesired phenomena associated with a native surface in contact with oneor more biomaterials. In other embodiments, it may be useful to enhancesurface properties of the material, device, and/or apparatus tointroduce a desired characteristic to the surface, thereby broadeningthe handling, manipulation or processing capabilities of the material,device, and/or apparatus. To that end, molecules which can modify asurface to either decrease undesired properties or introduce desirableproperties are needed.

A microfluidic device is described herein having an enclosure includinga base, a cover, and microfluidic circuit material defining a fluidiccircuit therein, where at least one inner surface of the base, the coverand the microfluidic circuit material has a first covalently boundsurface modification including a first linking group, and a firstmoiety, wherein the first moiety is a first surface contact moiety or afirst reactive moiety; wherein at least one inner surface of the base,the cover and the microfluidic circuit material has a second covalentlybound surface modification including a second linking group, and asecond moiety, wherein the second moiety is a second surface contactmoiety or second reactive moiety, and where the first linking group andthe second linking group are different from each other or the firstcovalently bound moiety is different from the second covalently boundmoiety. The first surface modification may be a covalently modifiedsurface and the second surface modification may be a functionalizedsurface. In other embodiments, the first surface modification may be afirst covalently modified surface and the second surface modificationmay be a second covalently modified surface having either a differentlinking group or different surface modifying ligand.

Modifying Reagent: Surface Modifying Compound.

In various embodiments, a surface modifying compound may include asurface modifying ligand which may be a non-polymeric moiety such as analkyl moiety, a substituted alkyl moiety, such as a fluoroalkyl moiety(including but not limited to a perfluoroalkyl moiety) or an alkyleneoxide moiety, amino acid moiety, alcohol moiety, amino moiety,carboxylic acid moiety, phosphonic acid moiety, sulfonic acid moiety,sulfamic acid moiety, or saccharide moiety covalently modifies thesurface to which it is attached. The surface modifying compound alsoincludes a connecting moiety, a group which covalently attaches thesurface modifying ligand to the surface, as shown schematically inEquation 1. Depending on the composition of the surface, the connectingmoiety may be a silicon containing moiety such as —Si(T)₂W, where W is-T, —SH, or —NH₂; and T is independently OH, OC₁₋₆alkyl, or halo, or acombination thereof; a phosphonic acid moiety or an activated formthereof, a maleimide moiety, a terminal olefin, or any suitableconnecting moiety known in the art. The surface modifying ligand isattached to the covalently modified surface via a linking group LG,which is the product of the reaction of the connecting moiety withfunctional groups of the surface (including hydroxide, oxide, amine orsulfur). A linking group LG may include a siloxy, phosphonate, alkylsulfide and the like. In some embodiments, the linking group LG may be asiloxy or phosphonate group.

In some embodiments, the surface modifying compound has a structure ofFormula XXXII:

V-L_(sm)-Surface modifying ligand  Formula XXXII;

wherein connecting moiety V is —P(O)(OH)₂ or —Si(T)₂W; W is -T, —SH, or—NH₂ and is the moiety configured to connect to the surface; eachinstance of T is independently OH, OC₁₋₆alkyl, or halo. L_(sm) is alinker including 1 to 200 non-hydrogen atoms selected from anycombination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorusatoms and further includes 0, 1, 2, 3, or 4 coupling groups CG. Thenumber of non-hydrogen atoms that form CG is not included in the size ofL_(sm), and is not limited by the size of L_(sm). The surface modifyingligand may include 0, 1, 2, or 3 CG.

In some embodiments, the surface modifying compound of Formula XXXII maybe a compound of Formula I:

V—(CH₂)_(n)-surface modifying ligand  Formula I;

wherein connecting moiety V is —P(O)(OH)Q- or —Si(T)₂W; W is -T, —SH, or—NH₂ and is the moiety configured to connect to the surface; Q is —OHand is the moiety configured to connect to the surface; and n is aninteger of about 3-21. In some embodiments, n is an integer of about 7to 21. Each instance of T is independently OH, OC₁₋₆alkyl, or halo,where alkyl includes but is not limited to methyl, ethyl, n-propyl,2-propyl, n-butyl, and the like. In some embodiments, T is OH,OC₁₋₃alkyl or Cl. The surface modifying ligand may include 0, 1, 2, or 3CG.

In some embodiments, the compound of Formula I is a compound having astructure of Formula II:

wherein W, T, and n are as defined above for Formula I. The surfacemodifying ligand may include 0, 1, 2, or 3 CG.

In other embodiments, the compound of Formula I is a compound of FormulaIII:

wherein R is C₁₋₆alkyl and n is an integer of 3-21. The surfacemodifying ligand may include 0, 1, 2, or 3 CG.

The surface modifying compound used to covalently modify a surface ofthe inner surface(s) of a microfluidic device, as described herein,introduces the surface modifying ligand having a surface contact moiety,which supports cell growth, viability or portability of biologicalcells. The surface modifying ligand including a surface contact moietycan include anionic, cationic, or zwitterionic moieties, or anycombination thereof. Without intending to be limited by theory, bypresenting cationic moieties, anionic moieties, and/or zwitterionicmoieties at the inner surfaces of an enclosure of the microfluidicdevice, the surface modifying ligand of the covalently modified surfacecan form strong hydrogen bonds with water molecules such that theresulting water of hydration acts as a layer (or “shield”) thatseparates the biological micro-objects from interactions withnon-biological molecules (e.g., the silicon and/or silicon oxide of thesubstrate). In addition, in embodiments in which the covalently modifiedsurface is used in conjunction with coating agents, the anions, cations,and/or zwitterions of the surface contact moiety of the surfacemodifying ligand can form ionic bonds with the charged portions ofnon-covalent coating agents (e.g. proteins in solution) that are presentin a medium (e.g. a coating solution and/or a fluidic medium forsupporting biological cells) in the enclosure. In other embodiments, thesurface modifying ligand may include at least one amino acid, which mayinclude more than one type of amino acid. Thus, the surface modifyingligand may include a peptide or a protein. In some embodiments, thesurface modifying ligand may include an amino acid which may provide azwitterionic surface to support cell growth, viability, portability, orany combination thereof.

In still other embodiments, the surface modifying ligand may present ahydrophilic surface contact moiety at its enclosure-facing terminus,including but not limited at least one alkylene oxide moiety. One usefulclass of alkylene ether containing polymers is polyethylene glycol (PEGM_(w)<100,000 Da). In some embodiments, a PEG may have an M_(w) of about100 Da, 300 Da, 500 Da, 1000 Da, or 5000 Da. In other embodiments, ahydrophilic surface modifying ligand may include one or moresaccharides. The covalently linked saccharides may be mono-, di-, orpolysaccharides. Like the charged moieties discussed above, thehydrophilic surface modifying ligand can form strong hydrogen bonds withwater molecules such that the resulting water of hydration acts as alayer (or “shield”) that separates the biological micro-objects frominteractions with non-biological molecules (e.g., the silicon and/orsilicon oxide of the substrate).

The surface modifying ligand may alternatively include one or more aminogroups as a surface contact moiety. The amino group may be a substitutedamine moiety, guanidine moiety, nitrogen-containing heterocyclic moietyor heteroaryl moiety. The amino containing moieties may have structurespermitting pH modification of the environment within a microfluidicdevice. In some embodiments of the microfluidic device described herein,the environment may be modified within pens opening to a flow region(which may be the same as or may be different from sequestration pens,as described herein), and/or flow regions (which may include channels).

In various embodiments, a surface modifying compound may include alinear backbone of 8 to 26 atoms, wherein the atoms are carbon, oxygen,nitrogen or sulfur; and a connecting moiety selected from —P(O)(OH)₂ and—Si(Y)₃, where Y is Cl, OC₁₋₃ alkyl, or OH, and non-backbonesubstituents of carbon atoms of the linear backbone are hydrogen orfluorine. The surface modifying compound can attach to functional groupson the surface (including hydroxide, oxide, amine or sulfur) through theconnecting moiety. A first end of the linear backbone is connected tothe connecting moiety through a bond to the phosphorus or silicon of theconnecting moiety and a second end of the linear backbone is distal toand not connected to the surface. Independently for each carbon of thelinear backbone, the non-backbone substituents are either all hydrogenor all fluorine. In some embodiments, the linear backbone may be allcarbon atoms. A linear backbone having all carbon backbone atoms mayhave non-backbone substituents that are all hydrogen atoms.

In some embodiments, the linear backbone of the surface modifyingcompound may be part of a linker L_(sm), as described above, and mayinclude two carbon atoms disposed at the first end of the linearbackbone (e.g., attached directly to the connecting moiety), and thenon-backbone substituents for each of the two carbons may be hydrogen.In some embodiments, the linear backbone may include a sulfur atom. Insome embodiments, the linear backbone may include two sulfur atoms, andthe two sulfur atoms are disposed adjacent to each other. When twosulfur atoms disposed adjacent to each other are present in the linearbackbone, then the two sulfur atoms are not disposed at the first end(e.g., neither of the two sulfur atoms are not directly connected to theconnection moiety) or the second end of the linear backbone (e.g.,located at the end of the modifying compound, distal to the connectionto the surface). In some embodiments, a disulfide moiety of the linearbackbone may be a cleavable motif, and may permit removal of part or allof the surface modifying ligand. Other cleavable motifs may be includedin the linker L_(sm) of the surface modifying compound, as describedherein.

In some embodiments, the surface modifying compound may contain 0, 1, 2,3 or 4 coupling groups CG as described herein. The surface modifyingcompound may have been formed from two or more portions coupled to eachother to provide the linking group and the surface modifying ligandwhere the CG may be part of linker L_(sm) or may be part of the surfacemodifying ligand (which also contains the surface contact moiety).

In some embodiments, the surface modifying compound may include carbonatoms forming a linear chain (e.g., a linear chain of at least 10carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be anunbranched alkyl moiety. In some embodiments, the alkyl group mayinclude a substituted alkyl group (e.g., some of the carbons in thealkyl group can be fluorinated or perfluorinated). In some embodiments,the alkyl group may include a first segment, which may include aperfluoroalkyl group, joined to a second segment, which may include anon-substituted alkyl group, where the first and second segments may bejoined directly or indirectly (e.g., by means of an ether linkage). Thefirst segment of the alkyl group may be located distal to the linkinggroup, and the second segment of the alkyl group may be located proximalto the connecting moiety.

In other embodiments of the surface modifying compound, the linearbackbone may include one or more oxygen atoms. Each of the one or moreoxygen atoms may not be connected directly to another oxygen, sulfur ornitrogen, and may not be disposed at the first end of the linearbackbone. In some embodiments, when the linear backbone includes one ormore oxygen atoms, each of the one or more oxygen atoms may not bedisposed at the second end of the linear backbone. In some embodiments,each of the one or more oxygen atoms may be disposed within the linearbackbone such that at least two backbone atoms adjacent to each oxygenatom proximal to the first end of the linear backbone are carbon atomscomprising hydrogen non-backbone substituents and at least two backboneatoms adjacent to each oxygen atom distal to the first end of the linearbackbone are carbons comprising hydrogen substituents.

A covalently bonded modification may be introduced to the surface uponreaction with the compound of Formula XXXII to provide a surface havinga structure of Formula XXXI:

where LG is —W—Si(OZ)₂O— or —OP(O)₂O—; W is O, S, or N, Z is a bond toan adjacent silicon atom or is a bond to the surface; L_(sm) is a linkercomprising 1 to 200 non-hydrogen atoms selected from any combination ofsilicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms andfurther comprises 0, 1, 2, 3, or 4 coupling groups CG; and

is the surface. In some embodiments, n is an integer of 7 to 21.

In some embodiments, the covalently bonded modification may have astructure of Formula VIII:

where W is O, S, or N; Z is a bond to an adjacent silicon atom or is abond to the surface; n is an integer of 3-21; and

is the surface. In some embodiments, W is O. In various embodiments, nis an integer of 7 to 21. The surface modifying ligand may include 0, 1,2, or 3 CG.

In other embodiments, the covalently bonded modification has a structureof Formula IX:

where n, and

are each defined as above. Z is a bond to an adjacent phosphorus atom oris a bond to the surface. The surface modifying ligand may include 0, 1,2, or 3 CG.

In some embodiments, the surface modifying ligand may have a structureof Formula X:

where L is a linker; and surface contact moiety is a moiety thatprovides improved contact characteristics for biological micro-objects,as described herein.

In other embodiments, the surface modifying ligand of the modifiedsurface may have a structure of Formula X:

where L is a linker; and surface contact moiety is a moiety thatprovides improved contact characteristics for biological micro-objects.

Linker L may be a bond or may include 1 to 200 non-hydrogen atomsselected from any combination of silicon, carbon, nitrogen, oxygen,sulfur and phosphorus atoms, subject to chemical bonding limitations asis known in the art. In some embodiments, linker L may include 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemicalbonding limitations as is known in the art. Linker L or the surfacecontact moiety may include 0, 1, or 3 coupling groups CG.

Coupling Group CG.

CG is a coupling group and may be any moiety such as but not limited totriazolylenyl, carboxamide, imide, ether, ester, keto, sulfonamide,sulfonate, cyclooctyl-fused diazine, alkene or aromatic moieties thatmay result from attaching the surface contact moiety to the remainder ofthe surface modifying reagent of Formula XXXII, or the surface modifyingcompound of Formula I, Formula II, or Formula III (e.g., formed as partof the synthesis of the surface modifying ligand).

In some other embodiments, CG is the moiety resultant from reaction ofthe reactive moiety of the functionalizing reagents of Formula XXXIII,Formula IV or Formula VI with a respective reaction pair moiety of asurface modifying reagent as described herein. For example, afunctionalizing reagent having a azide reactive moiety may form atriazolylenyl CG moiety upon forming a covalently modified surface ofFormula XXXI, Formula VIII, or Formula IX.

Coupling group CG may be a triazolylenyl moiety, which may be furthersubstituted, and may have one or more additional ring systems fused withthe triazolylenyl moiety. The additional fused ring system(s) may itselfbe further substituted with additional fused rings and may provide theattachment point to linker L-surface contact moiety. In someembodiments, the triazolylenyl moiety is fused with a cyclooctynyl ringsystem, which may be further substituted either with additional fusedrings, including but not limited to dibenzocylcooctynyl, or othersubstitutions such as fluorine (difluorinated cyclooctyne (DIFO)).

CG may in some embodiments be a noncovalent binding pair. For example,the noncovalent binding of biotin with streptavidin provides a verystable binding pair and may be a CG. Further, since streptavidin hasfour binding sites, two portions of a surface modifying ligand, surfacemodifying reagent, or functionalized surface may be joined by thesequence of biotin/streptavidin/biotin. For example, a functionalizedsurface has a biotin reactive moiety, streptavidin is then introduced tobind to the biotin reactive moiety, and finally where a secondbiotinylated moiety (such as biotin-fibronectin) is introduced and boundto another of the binding sites on streptavidin. The product is acovalently bound surface modification having a surface contact moiety offibronectin and the sequence of biotin/streptavidin/biotin is consideredto be a single coupling group CG. The streptavidin is performing therole of linking two similarly functionalized portions together.

Surface Contact Moiety.

The surface contact moiety of the surface modifying ligand may be anysurface contact moiety as described herein and in other portions of thedisclosure and may include non-polymeric or polymeric moieties. Thesurface contact moiety may include alkyl or fluoroalkyl (which includesperfluoroalkyl) moieties; mono- or polysaccharides (which may includebut is not limited to dextran); alcohols (including but not limited topropargyl alcohol); polyalcohols, including but not limited to polyvinylalcohol; alkylene ethers, including but not limited to polyethyleneglycol; polyelectrolytes (including but not limited to polyacrylic acidor polyvinyl phosphonic acid); amino groups (including derivativesthereof, such as, but not limited to alkylated amines, hydroxyalkylatedamino group, guanidinium, and heterocylic groups containing anunaromatized nitrogen ring atom, such as, but not limited to morpholinylor piperazinyl); carboxylic acids including but not limited to propiolicacid (which may provide a carboxylate anionic surface); phosphonicacids, including but not limited to ethynyl phosphonic acid (which mayprovide a phosphonate anionic surface); sulfonate anions;carboxybetaines; sulfobetaines; sulfamic acid; or amino acids. The alkylor perfluoroalkyl moieties may have a backbone chain length of greaterthan 10 carbons. In other embodiments, the surface contact moiety mayinclude saccharide moieties, and may be dextran. In other embodiments,the surface contact moiety may include alkylene ether moieties. Thealkylene ether moieties may be polyethylene glycol.

In various embodiments, the surface contact moiety may of the surfacemodifying ligand include non-polymeric moieties such as an alkyl moiety,a substituted alkyl moiety, such as a fluoroalkyl moiety (including butnot limited to a perfluoroalkyl moiety), amino acid moiety, alcoholmoiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety,sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety.Alternatively, the surface contact moiety may include polymericmoieties, which may be any of the moieties described above.

In some embodiments, the surface contact moiety may comprise carbonatoms forming a linear chain (e.g., a linear chain of at least 10carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be anunbranched alkyl moiety. In some embodiments, the alkyl group mayinclude a substituted alkyl group (e.g., some of the carbons in thealkyl group can be fluorinated or perfluorinated). In some embodiments,the alkyl group may include a first segment, which may include aperfluoroalkyl group, joined to a second segment, which may include anon-substituted alkyl group, where the first and second segments may bejoined directly or indirectly (e.g., by means of an ether linkage). Thefirst segment of the alkyl group may be located distal to the linkinggroup, and the second segment of the alkyl group may be located proximalto the linking group.

Cleavable Moiety.

The surface modifying ligand may further include a cleavable moiety,which may be located within the linker L_(sm) of the surface modifyingcompound, linker L of the surface modifying ligand or may be part of thesurface contact moiety of the surface modifying compound or surfacemodifying reagent. In some embodiments, a cleavable moiety may beincluded within linker Lm of the functionalized surface of Formula XXX,Formula V, or Formula VII. The cleavable moiety may be configured topermit disruption of the covalently modified surface. In someembodiments, disruption may be useful to promote portability of the oneor more biological cells after a period of culturing. The cleavablemoiety may be a photocleavable moiety such as nitro-substituted benzylesters (e.g., BroadPharm Catalog # BP-22675); a UV cleavable moiety suchas a substituted 1,2-diphenyl ethyl ketoester moiety (e.g., a benzilderivation such as BroadPharm Catalog # BP 22689); or may be a moietywhich can be cleaved under specific chemical conditions. For example, adisulfide linkage can be cleaved under conditions (e.g., reducingconditions such as dithiothreitol) that may not interfere with thegrowth or viability of the biological cells on the covalently modifiedsurface. Other useful cleavable moieties that may be incorporated withinsurface modifying ligands or functionalized surfaces can include avicinal diol moiety, which is cleavable by sodium periodate. The sodiumperiodate cleavage is another non-cytotoxic cleavage reagent. Diazomoieties, which are cleavable by dithionite, may also be a usefulcleavable moiety. Additionally, a 5, 5, dimethyl-exo-cyclohexen-yl-1,3,dione moiety may be a useful cleavable moiety for use in the surfacemodifying ligand or functionalized surface of Formula XXX, Formula V, orFormula VII, and may be cleaved by hydrazine solution.

Modifying Reagent: Surface Functionalizing Reagent.

A surface may be covalently modified by a functionalizing reagent, tointroduce a functionalized surface modification to one or more surfacesof the microfluidic device.

A functionalizing reagent is a compound of Formula XXXIII:

V-L_(fm)-R_(x)  Formula XXXIII;

wherein V is —P(O)(OH)₂ or —Si(T)₂W; W is -T, —SH, or —NH₂ and is themoiety configured to connect to the surface; T is independently OH,OC₁₋₆alkyl, or halo; L_(fm) is a linker comprising 1 to 200 non-hydrogenatoms selected from any combination of silicon, carbon, nitrogen,oxygen, sulfur and phosphorus atoms and further comprises 0, 1 or 2coupling groups CG; and R_(x) is a reactive moiety.

Reactive Moiety.

The reactive moiety may be any of an alkyne moiety, azide moiety, aminemoiety, carboxylic acid moiety, biotin moiety, streptavidin moiety,olefin moiety, trans cyclooctene moiety, s-tetrazine moiety, thiolmoiety, maleimide moiety, halide moiety, cyano moiety, isocyanatemoiety, epoxide moiety, hydroxyamine moiety, a masked hydroxyl such asacetate and the like, or sulfonyl fluoride moiety. This list of reactivemoieties is not limiting and any suitable reactive moiety may beselected for use with an appropriate reaction pair moiety. While mostreactive moieties react with a respective reaction pair moiety to form acovalently coupled CG, the high binding affinity between biotin andstreptavidin permits its use as a reactive moiety/reaction pair moiety.

The functionalized surface formed by the reaction of functionalizingreagent XXXIII has a structure of Formula XXX:

where LG is —W—Si(OZ)₂O— or —OP(O)₂O—; W is O, S, or N, Z is a bond toan adjacent silicon atom or is a bond to the surface, and L_(fm) andR_(x) are as defined for Formula XXXIII.

In some embodiments, a functionalizing reagent of Formula XXXIII may bea compound of Formula IV:

wherein R is OC₁₋₆alkyl and n is an integer of 3-21. Azide is thereactive moiety R_(x). In some embodiments of the compound of FormulaIV, n may be an integer of 7 to 21. For the compound of Formula IV, eachinstance of R may be independently chosen from H or C₁-C₆ alkyl, wherealkyl includes but not limited to methyl, ethyl, n-propyl, 2-propyl,n-butyl and the like. In some embodiments, R may be C₁-C₃ alkyl. In someembodiments, R may be methyl or ethyl. In various embodiments, each ofthe three instances of R is methyl or each of the three instances of Ris ethyl. In other embodiments, n may be 9, 14, or 16. In yet otherembodiments, n may be 9.

The functionalized surface formed by the reaction of the surface withthe surface functionalizing reagent of Formula IV may have a structureof Formula V:

wherein W is O, S, or N, Z is a bond to an adjacent silicon atom ofanother surface functionalizing ligand (—WSi(OZ)₂(CH₂)_(n)—N₃) alsobound to the surface or is a bond to the surface, n is an integer of3-21, and

is the surface. In some embodiments, n may be an integer of about 7 to21. In some embodiments, W may be O. In various embodiments, eachinstance of R may be independently chosen from H or C₁-C₆ alkyl, wherealkyl includes but not limited to methyl, ethyl, n-propyl, 2-propyl,n-butyl and the like. In some embodiments, R may be C₁-C₃ alkyl. In someembodiments, R may be methyl or ethyl. In various embodiments, each ofthe three instances of R is methyl or each of the three instances of Ris ethyl. In other embodiments, n may be an integer of 7 to 21. In someembodiments, n may be an integer of 9 to 21, 10 to 21, 11 to 21, 12 to21, 13 to 21, 14 to 21, 15 to 21, 16 to 21, 17 to 21, or 18 to 21. Inyet other embodiments, n may be an integer of 10 to 18, 12 to 18, 13 to18, or 14 to 18. In other embodiments, n may be 9, 14, or 16. In yetother embodiments, n may be 9.

In other embodiments, a surface functionalizing reagent of FormulaXXXIII may be a compound of Formula VI:

wherein n is an integer of 3 to 21 and each instance of R isindependently H or C₁-C₆ alkyl. Alkyne is the reactive moiety R_(x) ofFormula VI. In some embodiments, n may be an integer of about 7 to 21.In various embodiments, each instance of R may be independently chosenfrom H or C₁-C₆ alkyl, where alkyl includes but not limited to methyl,ethyl, n-propyl, 2-propyl, n-butyl and the like. In some embodiments, Rmay be C₁-C₃ alkyl. In some embodiments, R may be methyl or ethyl. Invarious embodiments, each of the three instances of R is methyl or eachof the three instances of R is ethyl. In some embodiments, n may be aninteger of 9 to 21, 10 to 21, 11 to 21, 12 to 21, 13 to 21, 14 to 21, 15to 21, 16 to 21, 17 to 21, or 18 to 21. In yet other embodiments, n maybe an integer of 10 to 18, 12 to 18, 13 to 18, or 14 to 18. In otherembodiments, n may be 9, 14, or 16. In yet other embodiments, n may be9.

The compound of Formula VI may be covalently coupled to a surface viareaction of the siloxane moiety with nucleophilic groups of the surface,providing a functionalized surface having a structure of Formula VII:

where W, Z, and n are defined as above for Formula V, and

is the surface.

Covalently modified surface formed from the functionalized surface. Oncethe surface functionalization reagent has been coupled to the surface,the reactive moiety of the resultant functionalized surface of FormulaXXX, Formula V or Formula VII may be reacted in turn with a surfacemodifying reagent having a reaction pair moiety selected to be asuitable reaction partner to the reactive moiety of the functionalizedsurface. The surface modifying reagent has a structure of Formula XII:

RP-L-surface contact moiety  Formula XII;

where RP is a reaction pair moiety; L is a linker and surface contactmoiety is a moiety that provides improved contact characteristics forbiological micro-objects. Linker L may be a bond or may include 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemicalbonding limitations as is known in the art. In some embodiments, linkerL may include 1 to 200 non-hydrogen atoms selected from any combinationof silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms,subject to chemical bonding limitations as is known in the art. Linker Lor the surface contact moiety may include 0, 1, 2, or 3 coupling groupsCG. Surface contact moiety is any surface contact moiety describedherein.

Reaction Pair Moiety.

The reaction pair moiety RP is a moiety that can react with the reactivemoiety of the functionalized surface. For example, a reactive moietyR_(x) may be alkyne and a corresponding reaction pair moiety RP may bean azide. Alternatively, R_(x) may be azide and RP may be alkyne. Otherpairs of reactive moiety R_(x):reaction pair moiety RP may include, butare not limited to cyano and azide; carboxylic acid and amine; olefinand nucleophile; amine and sulfonyl fluoride; trans cyclooctene ands-tetrazine, thiol and maleimide; halide and nucleophile; isocyanate andamines; epoxide and nucleophile; hydroxyamine and aldehyde or ester; anda masked hydroxyl such as acetate and nucleophile. A special case ofRx:RP pair is biotin and streptavidin as it is not a covalent pairingbut an extremely stable noncovalent binding pair that may be used as anR_(x):RP pair.

When the functionalized surface has an azide or a alkynyl moiety asR_(x), the surface modifying reagent has a reaction pair moiety RP whichis an alkyne or azide, respectively, which can react form atriazolylenyl moiety via a cyclization reaction (“Click reaction”) as isknown in the art. In some embodiments, the reactive moiety R_(x) or thereaction pair RP moiety is an acyclic alkyne. In other embodiments, thes the reactive moiety R_(x) or the reaction pair RP moiety is a cyclizedalkyne, which may be part of a cyclooctyne. In some embodiments, thecyclooctyne may be strained. The cyclooctyne may have further cyclicrings fused to the cyclooctyne, such as benzo group, and may be adibenzocyclooctyne. In other embodiments, the cyclooctyne may havefluoro substituents. When the alkyne of the surface modifying reagent isa cyclooctyne, the surface contact moiety of the reagent is attached tothe cyclooctyne via the linker L, which may be attached to any suitableposition on the cyclooctyne. When the alkyne of the functionalizedsurface is a cyclooctyne, the linking group attaching the cyclooctyne tothe surface is attached to the cyclooctyne at any suitable position onthe cyclooctyne.

The covalently modified surface resulting from the reaction of thefunctionalized surface of Formula XXX:

with a surface modifying reagent of Formula XII may have a structure ofFormula XXXI:

wherein LG, L_(sm), surface modifying ligand and

are as defined above, and L_(sm) or the surface modifying ligandincludes at least one CG, and may further have 2, 3, or 4 CG.

In some embodiments, the covalently modified surface formed from thefunctionalized surface of Formula XXXI, Formula V, or Formula VII mayhave a structure of Formula VIII:

where W, Z, n, and

are each defined as above. The surface modifying ligand may include 0,1, 2, or 3 CG.

In some embodiments, the covalently modified surface formed from thefunctionalized surface of Formula XXXI may have a structure of FormulaIX:

where Z, n, and

are each defined as above. The surface modifying ligand may include 0,1, 2, or 3 CG.

Additional Functionalization of the Functionalized Surface.

In yet other embodiments, the functionalized surface of Formula XXX mayhave a further portion of functionalization added by reaction with asecondary functionalizing reagent of Formula XXXIV:

RP-L_(fm)-R_(x2)  Formula XXXIV,

wherein RP is a reaction pair moiety for reacting with the reactivemoiety of Formula XXX; R_(x2) is a reactive moiety selected to not reactwith the reactive moiety of the functionalizing surface of Formula XXX;and, L_(fm) is a linker comprising 1 to 200 non-hydrogen atoms selectedfrom any combination of silicon, carbon, nitrogen, oxygen, sulfur andphosphorus atoms and further comprises 0, 1 or 2 coupling groups CG.R_(x2) is selected to have an orthogonal reaction pair moiety such thatit does not interfere with the coupling of RP to the R_(x) moiety of thefunctionalized surface. In some nonlimiting examples, when R_(x) of thefunctionalized surface is azide, R_(x2) may be selected to be amine,epoxide, or sulfonyl fluoride. This ability affords control in furtherelaboration of the functionalized surface.

The product is a functionalized surface of Formula XXXV, wherein thesecond functionalized surface comprises 1, 2, or 3 CG:

where R_(x2) is as defined for Formula XXXIV, and L_(fm) and LG are asdefined above for Formula XXX. When a functionalized surface of FormulaV or Formula VII is reacted with a secondary functionalizing reagent ofFormula XXXIV, the produce is a functionalized surface of formula XXXV,wherein LG is —W—Si(OZ)₂O— and W is O, S, or N. In some embodiments, Wis O.

The functionalized surface of Formula XXXV may be converted to acovalently modified surface of Formula XXXI:

where LG, L_(sm) and surface modifying ligand are defined as above, byfurther reaction with a surface modifying reagent of Formula XII. Inthis embodiment, the surface modification (e.g., covalently modifiedsurface) includes at least 2 CG within L_(sm).

FIG. 2H depicts a cross-sectional view of a microfluidic device 290comprising an exemplary covalently modified surface 298. As illustrated,the covalently modified surface 298 (shown schematically) can comprise amonolayer of densely-packed molecules covalently bound to both the innersurface 294 of the substrate 286 and the inner surface 292 of the cover288 of the microfluidic device 290. The covalently modified surface s298 can be disposed on substantially all inner surfaces 294, 292proximal to, and facing inwards towards, the enclosure 284 of themicrofluidic device 290, including, in some embodiments and as discussedabove, the surfaces of microfluidic circuit material (not shown) used todefine circuit elements and/or structures within the microfluidic device290. In alternate embodiments, the covalently modified surface 298 canbe disposed on only one or some of the inner surfaces of themicrofluidic device 290.

In the embodiment shown schematically in FIG. 2H, the covalentlymodified surface 298 includes a monolayer of substituted siloxanemolecules, each molecule covalently bonded to the inner surfaces 292,294 of the microfluidic device 290 via a siloxy linker 296. Forsimplicity, additional silicon oxide bonds are shown linking to adjacentsilicon atoms, but the invention is not so limited. In some embodiments,the surface modifying ligand 298 can include any kind of nonpolymericmolecule as described herein (e.g. a fluorinated alkyl group, apolyethylene glycol containing group, an alkyl group containing acarboxylic acid substituent) at its enclosure-facing terminus (i.e. theportion of the monolayer of the surface modifying ligand 298 that is notbound to the inner surfaces 292, 294 and is proximal to the enclosure284). While FIG. 2H is discussed as having non-polymeric surfacemodifying ligands, polymeric moieties may also be a suitable surfacecontacting moiety and/or surface modifying ligand, and be incorporatedinto the covalently modified surface, as described herein.

In other embodiments, the surface modifying ligand 298 used tocovalently modify the inner surface(s) 292, 294 of the microfluidicdevice 290 can include anionic, cationic, or zwitterionic moieties, orany combination thereof. Without intending to be limited by theory, bypresenting cationic moieties, anionic moieties, and/or zwitterionicmoieties at the inner surfaces of the enclosure 284 of the microfluidiccircuit 120, the surface modifying ligand of the covalently modifiedsurface 298 can form strong hydrogen bonds with water molecules suchthat the resulting water of hydration acts as a layer (or “shield”) thatseparates the biological micro-objects from interactions withnon-biological molecules (e.g., the silicon and/or silicon oxide of thesubstrate).

Surface to be Modified.

A surface capable of being modified by the compound of any of FormulaeXXXII, I, II, III, XXXIII, IV, VI, XII or XXXIV may be a metal, metaloxide, glass or polymer. Some materials that may have a covalentlymodified surface or a functionalized surface introduced therein in mayinclude but not be limited to silicon and its oxides, silicones,aluminum or its oxide thereof (Al₂O₃), Indium Tantalum Oxide (ITO),titanium dioxide (TiO₂), zirconium oxide (ZrO2), hafnium(IV) oxide(HfO₂), tantalum (V) oxide (Ta₂O₅), or any combination thereof. Polymersmay include any suitable polymer. A suitable polymer may include but isnot limited to (e.g. rubber, plastic, elastomer, silicone,organosilicone, such as polydimethylsiloxane (“PDMS”), or the like),which can be gas permeable. Other examples can include molded glass, apatternable material such as a silicone polymer (e.g. photo-patternablesilicone or “PPS”), photo-resist (e.g., an epoxy-based photo-resist suchas SU8), or the like. In other embodiments, a surface of a material suchas a natural fiber or wood may be modified by the compound of any ofFormulae XXXII, I, II, III, XXXIII, IV, VI, XII or XXXIV to introduce acovalently modified surface of Formula XXXI, Formula VIII, or Formula IXor a functionalized surface of Formula XXX, Formula V, Formula VII orFormula XXXV.

The surface to be modified may include a nucleophilic moiety includingbut not limited to hydroxide, amino and thiol. The nucleophilic moiety(e.g., hydroxide (in some embodiments referred to as oxide)) on thesurface may react with the compound of any of Formulae XXXII, I, II,III, XXXIII, IV, or VI to covalently link the compound to the surface,via a siloxy linking group or phosphonate linking group, to provide thefunctionalized surface. The surface to be modified may include nativenucleophilic moieties, or may be treated with reagents (e.g., piranhasolution) or by plasma treatment to introduce nucleophilic moieties(e.g., hydroxide (alternatively referred to as oxide)).

Physical and Performance Properties of the Covalently Modified Surface.

In some embodiments, the covalently modified surface of Formula XXXI,Formula VIII or Formula IX may have a thickness of less than 10 nm(e.g., less than about 7 nm, less than about 5 nm, or about 1.5 to 3.0nm). This may provide an advantageously thin layer on the modifiedsurface, particularly in contrast with other hydrophobic materials suchas CYTOP®, a perfluoro tetrahydrofuranyl polymer which is spin-coatedyielding a typical thickness of about 30 to 50 nm. Data shown in Table 1is for a silicon/silicon oxide native surface converted to afunctionalized surface (e.g., Formula XV (a specific member of the classof Formula V) or a surface modified with surface contact moieties (e.g.,Formula XVI and Formula XVII, specific embodiments of a modified surfaceof Formula VIII). Contact angle measurements were obtained using thestatic sessile drop method. (Drelich, J. Colloid Interface Sci. 179,37-50, 1996.) Thickness was measured by ellipsometry.

TABLE 1 Physical data for selected surfaces. Contact Angle (water orFunctionalized or Modified Surface aqueous solution) Thickness FormulaXV 80 degrees 1.4-1.5 nm See Examples 3 and 5 Formula XVI (PEG, ~5000Da) 35 degrees ~3 nm* See Example 6 Formula XVII (Dextran ~3000 Da) 40degrees Not See Example 7 available Formula XVIII (PEG, ~5000 Da) 34degrees ~4 nm* See Example 8 Formula XIX (PGA) 17 degrees ~5 nm SeeExample 9 Formula XX (biotin PEG) 39 degrees ~5 nm See Example 10Formula XXI (PC biotin PEG) 42 degrees ~5 nm See Example 11 Formula XXII(propiolic acid) 64 degrees 2 nm See Example 12 Formula XXIII (propargylamine) na na See Example 13 Formula XXIV (PEG carboxylic acid) 42degrees ~5 nm See Example 14 Formula XXV (poly lysine) 50 degrees 3 nmSee Example 15 Formula XXVI ((polyglutannic acid) 54 degrees 3 nm SeeExample 16 Formula XXVII (Biotin PEG with 66 degrees 2 nm disulfidelinkage) See Example 17As expected, modification of a silicon/silicon oxide surface to have afunctionalized surface of Formula XV, resulted in a modified surfacehaving an increased contact angle for water, of about 80 degrees. Thisis in contrast to the contact angle for water on a plasma cleanedsilicon surface of less than 10 degrees. Further elaboration of thefunctionalized surface to provide the modified surface of Formula XVI(including PEG moieties), yields a much more hydrophilic surface with adecreased contact angle of 35 degrees. A modified surface having astructure of Formula XVII (including dextran) had a contact angle of 40degrees.

Other analytical methods suitable to characterize the surface caninclude infrared spectroscopy and/or X-ray photoelectron spectroscopy.

In some embodiments, the modified surface of Formula XXXI, Formula VIII,or Formula IX may form a monolayer. The uniformity and evenness of amonolayer modified surface may provide advantageous performance,particularly if the monolayer modified surface has other functionalattributes. For example, the modified surface of Formula XXXI, FormulaVIII or Formula IX may also include an electrode activation substrate,and optionally further may include a dielectric layer, as may be foundin materials, devices and/or apparatuses having a dielectrophoresisconfiguration or an electrowetting configuration. The lack ofunsaturation of the perfluoroalkyl moieties of the modified surface canminimize “charge trapping” compared to a monolayer containing, forexample olefinic or aromatic moieties. Additionally, the densely-packednature of the monolayer formed in the surfaces of Formula XXXI, FormulaVIII or Formula IX may minimize the potential for cations to be driventhrough the monolayer to the underlying metal, metal oxide, glass orpolymer substrate. Without being limited by theory, the disruption ofthe substrate surface by addition of cations to substrate compositionmay disrupt the electrical properties of the substrate, thereby reducingits ability to function electrokinetically.

Further, the ability to introduce the modified surface via a covalentlinkage may increase the dielectric strength of the modified surface andprotect the underlying material from breakdown under application of anelectric field. The uniformity and thinness of a dielectrophoretic orelectrowetting surface of a material, device and/or apparatus having acovalently modified structure of Formula XXXI, Formula VIII or FormulaIX, may further provide advantageous benefit for such modifieddielectrophoretic and/or electrowetting surface when the material,device and/or apparatus is optically actuated.

In some embodiments, the modified surface does not require a perfectlyformed monolayer to be suitably functional for operation. The physicalthickness and uniformity of the layer in the surface of any of FormulaXXXI, Formula VIII, Formula IX, Formula XXX, Formula V, Formula VII orFormula XXXV can be measured using an ellipsometer.

Multiple Covalently Bonded Surface Modifications and MultilayerSurfaces.

The microfluidic device may have more than one region within themicrofluidic device having a covalently modified surface modificationwhere each region has only one kind of covalently linked moiety.Alternatively, the microfluidic device may include more than onedifferent kind of covalently linked moiety on a single selected surface(e.g., a common inner surface of the microfluidic device) or on all ofthe internal surfaces of the microfluidic device.

For example, a first covalently bonded surface modification of a surfacemay have a specified number of non-hydrogen atoms as part of the linkerand/or surface modifying ligand. A second covalently bonded surfacemodification of this surface may include a surface contact moiety havingone or more charged moieties covalently attached to a linker having agreater number of non-hydrogen atoms, which may provide capacity topresent the charged moieties further away from the surface so modified,potentially in closer contact with biological micro-objects within themicrofluidic environment.

In another instance, the modified surface may have a first covalentlybonded surface modification having a first type of less stericallydemanding surface contact moiety and fewer non-hydrogen atoms in thelinker attaching the first covalently bonded surface modification to thesurface. The modified surface may have a second covalently bondedsurface modification having a sterically demanding surface contactmoiety and a linker having a greater number of non-hydrogen atoms. Thismixture of covalently bonded surface modifications can help to presentthe sterically demanding surface contact moiety while prevent undesiredinteractions with silicon/silicon oxide, hafnium oxide or alumina makingup the surface itself. In another example, the covalently linkedmoieties may provide a zwitterionic surface presenting oppositelycharged surface contact moieties in a random fashion on the surface.

In other embodiments, the covalently modified surface may have increasedhydrophilic and/or amphiphilic characteristics by introducing acombination of a first covalently bonded surface modification and asecond covalently bonded surface modification. Introduction of thecombination of first and second covalently bonded surface modificationscan provide modulated or customizable hydrophilic, amphiphilic orhydrophobic characteristics to the surface (including a common innersurface of the microfluidic device.) The increased hydrophilic and/oramphiphilic character of a covalently modified surface may providehydrophilic functionalities and/or hydrophobic moieties to whichbiological micro-objects may associate without irreversibly adhering.These associations may provide a beneficial environment during cellculture compared to native, unmodified surfaces of a microfluidicdevice.

Each of these characteristics may increase the durability,functionality, and/or biocompatibility of the modified surface. Each ofthese characteristics may further benefit the viability (includinggrowth rate and/or cell doubling rate), nature of the colony formed upona covalently modified surface having a structure of Formula XXXI,Formula VIII or Formula IX. Improvement of viability may includeproviding surface contact moieties providing adherent cells withsuitable anchoring sites which provide sufficient mechanical resistanceto promote growth. The covalently modified surface of Formula XXXI,Formula VIII or Formula IX may improve portability (including viabilityupon export) of micro-objects or biomolecules upon the modified surfaceand within devices and/or apparatuses having a covalently modifiedsurface. In some other embodiments, a covalently modified surface havinga structure of Formula XXXI, Formula VIII or Formula IX may providesurface contact moieties which discourage motile cells from migrationout of a specific region of a microfluidic device (e.g., a sequestrationpen), thereby minimizing cell movement out of the selected region. Theportability of the cells may in this instance be inhibited, preventingself-propelled movement of cells from one sequestration pen to anotherand minimizing contamination from sequestration pen to sequestrationpen. However, modulation of such inhibitory effect may be obtained byselection of the ratio of different covalently bound surfacemodification to still be able to export from the sequestration pen at adesired timepoint using forces such as gravity or dielectrophoresis(which may be light actuated).

The combination of covalently bonded surface modifications may be anycombination of a covalently modified surface and/or a functionalizedsurface or secondary functionalized surface as described herein. Anycombination of the linking group, linker, reactive moiety and/or surfacecontact moiety may be selected for the microfluidic device having afirst and a second covalently bound surface modification where the firstand the second covalently bonded surface modification are different fromeach other. The first and the second covalently bonded surfacemodifications may be any of Formula XXX, Formula V, Formula VII, FormulaXXXI, Formula VIII, and/or Formula IX.

In some embodiments, the microfluidic device may have one or both of thefirst and the second covalently bonded surface modifications that arefunctionalized surfaces, for further modification by the user. Amicrofluidic device having one or two functionalized surfaces, differingin reactive moiety, linker, and/or linking group, may either be reactedwith a surface modifying reagent (e.g, a reagent of Formula XII) toprovide a covalently modified surface or may be further functionalizedby reaction with a secondary functionalizing reagent (e.g., a reagent ofFormula XXXIV) to provide a secondary functionalized surface. Orthogonalchemistries (e.g. reaction moieties and reaction pair moieties, andreaction conditions), as are known in the art, may be selected to permitselective reaction of one functionalized surface in the presence of asecond functionalized surface or in the presence of a covalentlymodified surface. In one nonlimiting example, when an alkyne is presentas a first reactive moiety (Rx or Rx2) of the first covalently bondedsurface modification, it is designed to react with an azide as areaction pair moiety. The second covalently bound surface modificationmay have a second reactive moiety selected to be an amine or carboxylicacid, which do not take part in a “Click” type reaction.

In some embodiments, a covalently modified surface may include acombination of: a functionalized surface of Formula XXX, Formula V orFormula VII; and, a first covalently bound surface modification ofFormula XXXI, Formula VIII or Formula IX. The combination of thefunctionalized surface and the first covalently bound surfacemodification of Formula XXXI, Formula VIII or Formula IX may be randomlydistributed upon the covalently modified surface. In other embodiments,the covalently modified surface may have a first region having thefunctionalized surface of Formula XXX, Formula V or Formula VII abuttinga second region including the first covalently bound surfacemodification of Formula XXXI, Formula VIII or Formula IX. In otherembodiments, the covalently modified surface may include a plurality ofregions having the first covalently bound surface modification ofFormula XXX, Formula VIII or Formula IX separated from each other by thefunctionalized surface of Formula XXXI, Formula V or Formula VII. In yetother embodiments, the covalently modified surface may have a pluralityof regions including the functionalized surface of Formula XXX, FormulaV or Formula VII separated from each other by the first covalently boundsurface modification of Formula XXXI, Formula VIII or Formula IX.

In other embodiments, the covalently modified surface may have acombination of: a first covalently bound surface modification of FormulaXXXI, Formula VIII or Formula IX; and a second covalently bound surfacemodification of Formula XXXI, Formula VIII or Formula IX, where thefirst and the second covalently bound surface modifications aredifferent. In some embodiments, the first and second covalently boundsurface modifications, which differ from each other, may be randomlydistributed on the covalently modified surface. In some otherembodiments, the covalently modified surface may have a first regionhaving the first covalently bound surface modification of Formula XXXI,Formula VIII or Formula IX which abuts a second region having the secondcovalently bound surface modification of Formula XXXI, Formula VIII orFormula IX. In yet other embodiments, the covalently modified surfacemay have a plurality of regions having the first covalently boundsurface modification of Formula XXXI, Formula VIII or Formula IX, whichare separated from each other by the second covalently bound surfacemodification of Formula XXXI, Formula VIII or Formula IX

In further embodiments, the covalently modified surface may have acombination of: a first covalently bound surface modification of FormulaXXX, Formula V or Formula VII; and a second covalently bound surfacemodification of Formula XXX, Formula V or Formula VII, wherein the firstand the second covalently bound surface modifications are different andthe reactive moiety of the first covalently bound surface modificationdoes not react with the reactive moiety of the second covalently boundsurface modification. In some embodiments, the first and the secondcovalently bound surface modifications may be randomly distributed uponthe surface. In some other embodiments, the covalently modified surfacemay include a first region having the first covalently bound surfacemodification of Formula XXX, Formula V or Formula VII abutting a secondregion having the second covalently bound surface modification ofFormula XXX, Formula V or Formula VII. In yet other embodiments, thecovalently modified surface may have a plurality of regions includingthe first covalently bound surface modification of Formula XXX, FormulaV or Formula VII which are separated from each other by the secondcovalently bound surface modification of Formula XXX, Formula V orFormula VII.

Multiple Surface Modifications to Modulate Adhesion.

In some embodiments, it can be useful to modulate the capacity for cellsto adhere to surfaces within the microfluidic device. A surface that hassubstantially hydrophilic character may not provide anchoring points forcells requiring mechanical stress of adherence to grow and expandappropriately. A surface that presents an excess of such anchoringmoieties may prevent successfully growing adherent cells from beingexported from within a sequestration pen and out of the microfluidicdevice. combine surface second covalently bound surface modificationcomprises surface contact moieties to help anchoring adherent cells. Thestructures of the surfaces described herein and the methods of preparingthem provide the ability to select the amount of anchoring moieties thatmay be desirable for a particular use. It has been surprisinglydiscovered that a very small percentage of adherent type motifs may beneeded to provide a sufficiently adhesion enhancing environment. In someembodiments, the adhesion enhancing moieties are prepared before cellsare introduced to the microfluidic device. Alternatively, an adhesionenhancing modified surface may be provided before introducing cells, anda further addition of another adhesion enhancing moiety may be made,which is designed to attach to the first modified surface eithercovalently or non-covalently (e.g., as in the base ofbiotin/streptavidin binding)

In some embodiments, adhesion enhancing surface modifications may modifythe surface in a random pattern of individual molecules of a surfacemodifying ligand. In some other embodiments, a more concentrated patternof adhesion enhancing surface modifications may be introduced by usingpolymers containing multiple adhesion enhancing motifs such aspositively charged lysine side chains, which can create small regions ofsurface modification surrounded by the remainder of the surface, whichmay have hydrophilic surface modifications to modulate the adhesionenhancement. This may be further elaborated by use of dendriticpolymers, having multiple adhesion enhancing ligands. A dendriticpolymer type surface modifying compound or reagent may be present in avery small proportion relative to a second surface modification havingonly hydrophilic surface contact moieties, while still providingadhesion enhancement. Further a dendritic polymer type surface modifyingcompound or reagent may itself have a mixed set of end functionalitieswhich can additionally modulate the behavior of the overall surface.

In some embodiments, it may be desirable to provide regioselectiveintroduction of surfaces. It may be desirable to provide a first type ofsurface within the microfluidic channel while providing a surface withinthe sequestration pens opening off of the channel that provides theability to both culture adherent-type cells successfully as well aseasily export them using dielectrophoretic forces when desired. In someembodiments, the adhesion enhancing modifications may include cleavablemoieties. The cleavable moieties may be cleavable under conditionscompatible with the cells being cultured within, such that at anydesired timepoint, the cleavable moiety may be cleaved and the nature ofthe surface may alter to be less enhancing for adhesion. The underlyingcleaved surface may be usefully non-fouling such that export is enhancedat that time. While the examples discussed herein focus on modulatingadhesion and motility, the use of these regioselectively modifiedsurfaces are not so limited. Different surface modifications for anykind of benefit for cells being cultured therein may be incorporatedinto the surface having a first and a second surface modificationaccording to the disclosure.

Adherent Motifs.

Generally, a surface modification having a positively charged surfacecontact moiety such as poly-L-lysine, amine and the like may be usedwithin the modified surfaces of the disclosure. Another motif that maybe used includes the tripeptide sequence RGD, which is available as abiotinylated reagent and is easily adaptable to the methods describedherein. Other larger biomolecules that may be used include fibronectin,laminin or collagen, amongst others. Surprisingly, a surfacemodification having a structure of Formula XXVI, including apolyglutamic acid surface contact moiety, demonstrated the ability toinduce adherent cells to attach and grow viably. Another motif that mayassist in providing an adherent site is an Elastin Like Peptide (ELP),which includes a repeat sequence of VPGXG, where X is a variable aminoacid which can modulate the effects of the motif.

Regioselective Introduction of Differing Surfaces.

In some embodiments, a surface of the flow region (e.g., microfluidicchannel) may be modified with a first covalently bound surfacemodification and a surface of the at least one sequestration pen may bemodified with a second covalently bound surface modification, whereinthe first and the second covalently bound surface modification havedifferent surface contact moieties, different reactive moieties, or acombination thereof. The first and the second covalently bound surfacemodifications may be selected from any of Formula XXX, Formula V,Formula VII, Formula XXXI, Formula VIII, and/or Formula IX. When thefirst and the second covalently bound surface modifications both includefunctionalized surface of Formula XXX, Formula V, or Formula VII, thenorthogonal reaction chemistries are selected for the choice of the firstreactive moiety and the second reactive moiety. In various embodiments,all the surfaces of the flow region may be modified with the firstcovalent surface modification and all the surfaces of the at least onesequestration pen may be modified with the second covalent modification.

In some embodiments, the microfluidic device may have a surface of acombination of first and second covalently bound surface modificationselected from the surfaces of Formula V, Formula XVI, Formula XVII,Formula XVIII, Formula XIX, Formula XX, Formula XXI, Formula XXII,Formula XXIII, Formula XXIV, Formula XXV, Formula XXVI, Formula XXVII,Formula XXVIII, Formula XXIX, Formula XXXVI, Formula XXXVII, FormulaXXXVIII, Formula XXXIX, or Formula XL. In other embodiments themicrofluidic device may have one region of the microfluidic devicehaving a first covalently bound surface modification as well as a secondregion of the microfluidic device having a second covalently boundsurface modification (e.g., the flow region having the first covalentlybound surface modification and the sequestration pen having the secondcovalently bound surface modification) which may be selected from thesurfaces of Formula V, Formula XVI, Formula XVII, Formula XVIII, FormulaXIX, Formula XX, Formula XXI, Formula XXII, Formula XXIII, Formula XXIV,Formula XXV, Formula XXVI, Formula XXVII, [Formula XXVIII, Formula XXIX,Formula XXXVI, Formula XXXVII, Formula XXXVIII, Formula XXXIX, orFormula XL.

Methods of Preparation of the Covalently Modified Surface.

A surface of a material that may be used as a component of a device orapparatus may be modified before assembly of the device or apparatus.Alternatively, partially or completely constructed device or apparatusmay be modified such that all surfaces that will contact biomaterialsincluding biomolecules and/or micro-objects (which may includebiological micro-objects) are modified at the same time. In someembodiments, the entire interior of a device and/or apparatus may bemodified, even if there are differing materials at different surfaceswithin the device and/or apparatus. In some embodiments, the partiallyor completely constructed device and/or apparatus may be a microfluidicdevice as described herein, or a component thereof.

The surface to be modified may be cleaned before modification to ensurethat the nucleophilic moieties on the surface are freely available forreaction, e.g., not covered by oils or adhesives. Cleaning may beaccomplished by any suitable method including treatment with solventsincluding alcohols or acetone, sonication, steam cleaning and the like.Alternatively, or in addition, such pre-cleaning can include treatingthe cover, the microfluidic circuit material, and/or the substrate in anoxygen plasma cleaner, which can remove various impurities, while at thesame time introducing an oxidized surface (e.g. oxides at the surface,which may be covalently modified as described herein). Alternatively,liquid-phase treatments, such as a mixture of hydrochloric acid andhydrogen peroxide or a mixture of sulfuric acid and hydrogen peroxide(e.g., piranha solution, which may have a ratio of sulfuric acid tohydrogen peroxide from about 3:1 to about 7:1) may be used in place ofan oxygen plasma cleaner.

This can advantageously provide more sites for modification on thesurface, thereby providing a more closely packed modified surface layer.

Methods of Covalently Modifying a Surface Include Modifying a Surfacewith a Surface Modification Reagent of Formula XXXII, Formula I orFormula III.

Introducing a covalently modified surface may include contacting thesurface with the surface modifying compound of Formula XXXII:

V-L_(sm)-surface modifying ligand  Formula XXXII;

where V, L_(sm), and surface modifying ligand are defined as above;reacting the reagent of Formula XXXII with a nucleophilic moiety of thesurface; and, forming a covalently modified surface of Formula XXXI:

where LG. In some embodiments, the surface modifying compound of FormulaXXXII is a compound o and the surface

is defined as above f Formula I or Formula III:

where the covalently modified surface produced is a surface of FormulaVIII:

where Z is a bond to an adjacent silicon atom or is a bond to thesurface and the surface

is defined as above.

In other embodiments, the surface produced by reaction of the surfacemodifying compound of Formula XXXI is a surface having the structure ofFormula IX:

wherein Z is a bond to an adjacent phosphorus atom or is a bond to thesurface and the surface

is defined as above.

Methods of Covalently Modifying a Surface Include Functionalizing aSurface with a Functionalizing Reagent of Formula XXXIII, Formula IV orFormula VI.

Covalently functionalizing a surface with a functionalizing reagent ofFormula XXXIII may include: contacting the surface with the reagent ofFormula XXXIII:

V-L_(fm)-R_(x)  Formula XXXIII;

reacting the reagent of Formula XXXIII with a nucleophilic moiety of thesurface; and, forming a functionalized surface of Formula XXX:

wherein V, L_(fm), R_(x) and LG are as defined above.

In some embodiments, the functionalizing reagent of Formula XXXIII is afunctionalizing reagent of Formula IV or Formula VI, having a structureof one of the following formulae:

and providing a functionalized surface of Formula V or Formula VII,respectively:

W, Z, and n are as defined above, and

is the surface. In some embodiments, W is O. Each instance of R may beindependently H or C₁-C₆ alkyl. In some embodiments, n may be an integerof 7 to 21. In other embodiments, n may be 9, 14, or 16. In otherembodiments, n is 9. In some embodiments, R is C₁-C₃ alkyl. In otherembodiments, R is methyl or ethyl. In yet other embodiments, R ismethyl.

For Surface Modifying Reactions and Surface Functionalizing Reactions.

In some embodiments, the nucleophilic moiety of the surface is ahydroxide, amino or thiol. In some other embodiments, the nucleophilicmoiety of the surface may be a hydroxide. The surface may be a metal,metal oxide, glass, polymer, or any combination thereof. Surfacematerials that may be modified by this method may be any materialdescribed herein.

The contacting step may be performed by contacting the surface with aliquid solution containing the modifying reagent(s) of Formula XXXIII,Formula IV, Formula VI, Formula XXXII, Formula I, and/or Formula III,which may be any combination as described herein. For example, surfacesmay be exposed to solutions containing 0.01 mM, 0.1 mM, 0.5 mM, 1 mM, 10mM, or 100 mM of the modifying reagent(s) of Formula XXXIII, Formula IV,Formula VI, Formula XXXII, Formula I, and/or Formula III. The reactionmay be performed at ambient temperature and may be carried out for aperiod of time in the range of about 2 h, 4 h, 8 h, 12 h, 18 h, 24 h, orany value inbetween. Examples of solvents include but are not limitedto: dimethyl formamide (DMF), acetonitrile (ACN), toluene, 1,3bistrifluorobenzene, or Fluorinert™ (3M) fluorinated solvents. An acidsuch as acetic acid may be added to the solution to increase thereaction rate by promoting hydrolysis of the trialkoxy groups, ifpresent.

Alternatively, the surface may be contacted with a vapor phasecontaining the modifying reagent(s) of Formula XXXIII, Formula IV,Formula VI, Formula XXXII, Formula I, and/or Formula III, which may beany combination as described herein. In some embodiments, when thereacting step is performed by contacting the surface with the modifyingreagent(s) of Formula XXXIII, Formula IV, Formula VI, Formula XXXII,Formula I, and/or Formula III in the vapor phase, a controlled amount ofwater vapor is also present. The controlled amount of water vapor may beprovided by placing a preselected amount of magnesium sulfateheptahydrate in the same chamber or enclosure with the object having thesurface to be modified. In other embodiments, a controlled amount ofwater may be introduced into the reaction chamber or enclosure via anexternal water vapor feed. The reaction may take place under reducedpressure, relative to atmospheric pressure.

The reaction may be conducted at a temperature greater than about 95°C., or from about 100° C. to about 200° C. In various embodiments, thereaction may be conducted at a temperature of about 100° C., 110° C.,120° C., 130° C., 140° C., 150° C., 160° C., 170° C., 180° C., 190° C.,or about 200° C. The reaction may be permitted to continue for about 2h, 6 h, 8 h, 18 h, 24 h, 48 h, 72 h, 84 h, or more.

The modified and/or functionalized surface, in some embodiments, may bea monolayer. In some embodiments, the modified and/or functionalizedsurface may include at least one surface of a microfluidic circuitelement of a microfluidic chip. In other embodiments, the modifiedand/or functionalized surface may include all of the surfaces facingfluid bearing portions of a microfluidic device. For example, inexemplary microfluidic devices 200, 230, the inner surface of the topelectrode 210, the inner surface 208 of the electrode activationsubstrate 206, the surfaces of the microfluidic circuit material 116(See FIGS. 1B, 2A, 2B), all of which face the microfluidic channel 122and pens 224, 226, 228 may be functionalized. Similarly, in FIGS. 2D-2F,the inner surfaces of microfluidic circuit material 260, surfaces ofisolation structures 272 which define the sequestration pen 270, or allthe surfaces facing the microfluidic circuit 262 may be modified byreaction with the modifying reagent(s) of Formula XXXIII, Formula IV,Formula VI, Formula XXXII, Formula I, and/or Formula III.

Further Modification of a Functionalized Surface.

A method of covalently modifying a surface can include providing afunctionalized surface having a structure of Formula XXX:

where LG, L_(fm), and R_(x) are each defined as above and

is the surface; reacting the reactive moiety R_(x) with a surfacemodifying reagent having a structure of Formula XII:

RP-L-surface contact moiety  Formula XII;

where RP is a reaction pair moiety; L is a linker and surface contactmoiety is as defined above. Linker L may be a bond or may include 1 to200 non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemicalbonding limitations as is known in the art. In some embodiments, linkerL may include 1 to 200 non-hydrogen atoms selected from any combinationof silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms,subject to chemical bonding limitations as is known in the art. Linker Lor the surface contact moiety may include 0, 1, 2, or 3 coupling groupsCG; and thereby produces the covalently modified surface, having astructure of Formula XXXI:

where L_(sm) is as defined above.

In some embodiments, the functionalized surface of Formula XXX may be afunctionalized surface of Formula V or Formula VII:

where W is O, S, or N, Z is a bond to an adjacent silicon atom bound tothe surface or is a bond to the surface, n is an integer of about 3-21,In some embodiments, n is an integer of 7 to 21. The adjacent siliconatom to which Z is attached to may be incorporated in another surfacemodification molecule as described above. The covalently modifiedsurface produced may a surface modification molecule having a structureof Formula VIII:

where S, Z, n and

are each as defined above for Formula V or Formula VII. When Z is a bondto an adjacent silicon atom, the silicon atom may be part of anothersurface modification molecule of the following formula:

In some embodiments, n is an integer of 9 to 21. In other embodiments, nis 9, 14, or 16. In other embodiments, n is 9. In some embodiments, W isO.

In other embodiments, the product covalently modified surface having astructure of Formula XXXI, may have a structure of Formula IX:

wherein Z is a bond to an adjacent phosphorus atom or is a bond to thesurface and the surface

is defined as above.

When an alkyne is present in the functionalized surface (R_(x)) or thesurface modifying reagent of Formula XII (RP reaction pair moiety), itmay be an acyclic alkyne, and the reaction with an azide in a “Click”cyclization reaction may be catalyzed by a copper (I) salt. When acopper (I) salt is used to catalyze the reaction, the reaction mixturemay optionally include other reagents which can enhance the rate orextent of reaction. When an alkyne of the surface modifying reagent orthe functionalized surface is a cyclooctyne, the “Click” cyclizationreaction with an azide of the corresponding functionalized surface orthe surface modifying reagent may be copper free. A “Click” cyclizationreaction, thereby couples the surface modifying ligand to thefunctionalized surface to form the covalently modified surface. Thecyclization reaction may be catalyzed by a copper (I) salt, and mayoptionally include other reagents which can enhance the rate or extentof reaction. As described above for the functionalized surface, acovalently modified surface may be at least one surface of amicrofluidic device. In some embodiments, the covalently modifiedsurface may include substantially all the fluid-facing surfaces of theinterior of the microfluidic device.

Copper Catalysts.

Any suitable copper (I) catalyst may be used. In some embodiments,copper(I) iodide, copper (I) chloride, copper (I) bromide or anothercopper (I) salt. In other embodiments, a copper (II) salt may be used incombination with a reducing agent such as ascorbate to generate a copper(I) species in situ. Copper sulfate or copper acetate are non-limitingexamples of a suitable copper (II) salt. In other embodiments, areducing agent such as ascorbate may be present in combination with acopper (I) salt to ensure sufficient copper(I) species during the courseof the reaction. Copper metal may be used to provide Cu(I) species in aredox reaction also producing Cu(II) species. Coordination complexes ofcopper such as [CuBr(PPh3)3], silicotungstate complexes of copper,[Cu(CH3CN)4]PF6, or (Eto)3P CuI may be used. In yet other embodiments,silica supported copper catalyst, copper nanoclusters or copper/cuprousoxide nanoparticles may be employed as the catalyst.

Other Reaction Enhancers.

As described above, reducing agents such as sodium ascorbate may be usedto permit copper(I) species to be maintained throughout the reaction,even if oxygen is not rigorously excluded from the reaction. Otherauxiliary ligands may be included in the reaction mixture, to stabilizethe copper(I) species. Triazolyl containing ligands can be used,including but not limited to tris(benzyl-1H-1,2, 3-triazol-4-yl)methylamine (TBTA) or 3 [tris(3-hydroxypropyltriazolylmethyl)amine(THPTA). Another class of auxiliary ligand that can be used tofacilitate reaction is a sulfonated bathophenanthroline, which is watersoluble, as well, and can be used when oxygen can be excluded.

Other chemical couplings as is known in the art may be used to couple asurface modifying reagent to the functionalized surface as described forReaction Pair moiety.

Solvents and Reaction Conditions.

When an interior surface of a microfluidic device is the functionalizedsurface that reacts with a surface modifying reagent, the reaction maybe performed by flowing a solution of the surface modifying reagent intoand through the microfluidic device. In various embodiments, the surfacemodifying reagent solution may be an aqueous solution. Other usefulsolvents include aqueous dimethyl sulfoxide (DMSO), DMF, acetonitrile,or an alcohol may be used. The reaction may be performed at roomtemperature or at elevated temperatures. In some embodiments, thereaction is performed at a temperature in a range from about 15° C. toabout 60° C.; about 15° C. to about 55° C.; about 15° C. to about 50°C.; about 20° C. to about 45° C. In some embodiments, the reaction toconvert a functionalized surface of a microfluidic device to acovalently modified surface is performed at a temperature of about 15°C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., orabout 60° C.

Methods of Producing the Combined Surfaces.

A method of preparing a covalently modified surface on at least oneinner surface of a microfluidic device having an enclosure including abase, a cover and microfluidic circuit material defining a fluidiccircuit therein, includes: contacting the at least one inner surfacewith a first modifying reagent and a second modifying reagent; reactingthe first modifying reagent with a first nucleophilic moiety of the atleast one inner surface; reacting the second modifying reagent with asecond nucleophilic moiety of the at least one inner surface; and,forming the at least one covalently modified surface comprising a firstcovalently bound surface modification comprising a first linking groupand a first moiety that is a first surface contact moiety or a firstreactive moiety; and a second covalently bound surface modificationcomprising a second linking group and a second moiety that is a secondsurface contact moiety or second reactive moiety, wherein the firstlinking group is different from the second linking group or the firstmoiety is different from the second moiety.

In some embodiments, the reaction of the first modifying reagent withthe surface may be performed at the same time as reacting the secondmodifying reagent. For example, when the first modifying reagent and thesecond modifying reagent are both surface modifying compounds (e.g.,Formula XXXII, Formula I, Formula II, Formula III), a mixture of the twosurface modifying reagents, such as, but not limited to two differentsiloxane reagents, may be reacted via chemical vapor deposition at thesame time. The ratio of the two reagents may be varied in order toobtain different percentages of the two surface modifications (e.g.,surface modification ligands) as desired. In another example, thesurface may be a functionalized surface and the first and secondmodifying reagents are surface modifying reagent(s) (e.g, Formula XII)and/or secondary functionalizing reagent(s) (Formula XXXIV), and themixture of the two modifying reagents may be reacted with the reactivemoiety of the functionalized surface at the same time.

In other embodiments, the reaction of the first modifying reagent withthe surface may be performed before or after reacting the secondmodifying reagent with the at least one inner surface of themicrofluidic device. For example, the surface may be a functionalizedsurface (having a surface of Formula XXX, Formula V or Formula VII) andthe first modifying reagent may be a secondary functionalizing reagent,which can introduce an orthogonal R_(x2) or a surface modifying reagent.A reaction may be performed with limited amounts of the secondaryfunctionalizing reagent such that only a portion of the reactivemoieties R_(x) of the functionalized surface. Alternatively, the firstreaction may be performed with a limited amount of a surface modifyingreagent such that not all of the reactive moieties are coupled. This maybe performed to introduce, for example, a longer linker region in thesefirst introduced surface modifications. A following reaction canintroduce a second surface modification with use of a surface modifyingreagent that can introduce a desired surface contact moiety on all ofthe exposed reactive moieties or may only react with the unreactedoriginal reactive moiety sites. If an orthogonal R_(x2) has beenintroduced, a further reaction may be performed with a suitable surfacemodifying reagent which reacts only with R_(x2) and not with thereactive moiety R_(x) of the original functionalized surface.

In some embodiments, the reaction of the first modifying reagent and thereaction of the second modifying reagent with the surface may occur atrandom locations upon the surface. In other embodiments, the reaction ofthe first modifying reagent may occurs within a first region of thesurface and reaction of the second modifying reagent may occur within asecond regions of the surface abutting the first region. For example,the surfaces within the channel of the microfluidic device may beselectively modified with a first surface modification and the surfaceswithin the sequestration pen, which abut the surfaces within thechannel, may be selectively modified with a second, different surfacemodification.

In yet other embodiments, the reaction of the first modifying reagentmay occurs within a plurality of first regions separated from each otheron the at least one surface, and the reaction of the second modifyingreaction may occur at a second region surrounding the plurality of firstregions separated from each other.

In various embodiments, modification of one or more surfaces of themicrofluidic device to introduce a combination of a first surfacemodification and a second surface modification may be performed afterthe microfluidic device has been assembled. For one nonlimiting example,the first and second surface modification may be introduced by chemicalvapor deposition after assembly of the microfluidic device. In anothernonlimiting example, a functionalized surface having a first surfacemodification having a first reactive moiety and a second surfacemodification having a second, orthogonal reactive moiety may beintroduced. Differential conversion to two different surface modifyingligands having two different surface contact moieties can follow. Inanother embodiment, the microfluidic device may have a singlefunctionalized surface of Formula XXX, Formula V or Formula VII, whichmay be differentially modified by a mixture of two surface modifyingreagents, or a mixture of a surface modifying reagent and a secondaryfunctionalizing reagent (followed by conversion of the secondaryfunctionalized surface to a surface modifying ligand having a second,different surface contact moiety.

In some embodiments, at least one of the combination of first and secondsurface modification may be performed before assembly of themicrofluidic device. In some embodiments, modifying the at least onesurface may be performed after assembly of the microfluidic device.

In some embodiments, the method of preparing a microfluidic deviceincludes forming a first modified surface of one of the base or thecover before assembly of the microfluidic device; assembling themicrofluidic device, wherein assembling comprises assembling the firstcovalently modified surface of one of the base or the cover with themicrofluidic circuit materials and the other unmodified one of the coveror base; and forming a second modified surface on an unmodified surfaceof the assembled microfluidic device. For example, a first surfacemodification may be introduced on portions of the cover of themicrofluidic device, before assembly, and there may be unreactedportions of the cover still remaining. The microfluidic device may beassembled, and then reacted with a second surface modification (e.g., asurface modifying compound of Formula XXXII, Formula I, Formula II,Formula III) which not only reacts with all of the unmodified regionsremaining on the inner surface of the cover, but also reacts with all ofthe remaining interior surfaces of the base and microfluidic circuitmaterials.

In some embodiments, the covalently modified surface has a combinationof: a functionalized surface of Formula XXX, Formula V or Formula VII;and first covalently modified surface of Formula XXXI, Formula VIII orFormula IX disposed therein. The method may further include reacting thefunctionalized surface of Formula XXX, Formula V or Formula VII with asecondary functionalizing reagent of Formula XXXIV:

RP-L_(fm)-R_(x2)  Formula XXXIV,

In the presence of the first covalently modified surface of FormulaXXXI, Formula VIII or Formula IX, and producing a secondaryfunctionalized surface of Formula XXX. The method may further includereacting the secondary functionalized surface of Formula XXX, with asurface modifying reagent, having a structure of Formula XII, therebyproducing a second covalently modified surface of Formula XXXI, FormulaVIII or Formula IX in the presence of the first covalently modifiedsurface of Formula XXXI, Formula VIII or Formula IX.

Alternatively, the method may further include reacting the first formedfunctionalized surface of Formula XXX, Formula V or Formula VII with asurface modifying reagent, having a structure of Formula XII, therebyproducing a second covalently modified surface of Formula XXXI, FormulaVIII or Formula IX in the presence of the first covalently modifiedsurface of Formula XXXI, Formula VIII or Formula IX.

Uses.

Materials, devices and/or apparatuses having one or more surfacessuitable for modification to introduce a surface having a structure ofFormula XXXI, Formula VIII or Formula IX, as described above, mayinclude but are not limited to flow cytometry cells, apheresiscentrifugation equipment, tubing and receiving containers; ormicrofluidic devices handling cells, cell fragments, proteins, ornucleic acids for any kind of bioanalytical process or biomaterialsorting processes. Surfaces having a structure of Formula XXXI, FormulaVIII or Formula IX are not limited to micro-scale materials, devicesand/or apparatuses, but may be used for macroscale bioproductionequipment, medical devices, or water purification equipment andanalytical instrumentation thereof, for a few non-limiting examples.

Methods of Loading.

Loading of biological micro-objects or micro-objects such as, but notlimited to, beads, can involve the use of fluid flow, gravity, adielectrophoresis (DEP) force, electrowetting, a magnetic force, or anycombination thereof as described herein. The DEP force can be generatedoptically, such as by an optoelectronic tweezers (OET) configurationand/or electrically, such as by activation of electrodes/electroderegions in a temporal/spatial pattern. Similarly, electrowetting forcemay be provided optically, such as by an opto-electro wetting (OEW)configuration and/or electrically, such as by activation ofelectrodes/electrode regions in a temporal spatial pattern.

Microfluidic Devices and Systems for Operating and Observing SuchDevices.

FIG. 1A illustrates an example of a microfluidic device 100 and a system150 which can be used for maintaining, isolating, assaying or culturingbiological micro-objects. A perspective view of the microfluidic device100 is shown having a partial cut-away of its cover 110 to provide apartial view into the microfluidic device 100. The microfluidic device100 generally comprises a microfluidic circuit 120 comprising a flowpath 106 through which a fluidic medium 180 can flow, optionallycarrying one or more micro-objects (not shown) into and/or through themicrofluidic circuit 120. Although a single microfluidic circuit 120 isillustrated in FIG. 1A, suitable microfluidic devices can include aplurality (e.g., 2 or 3) of such microfluidic circuits. Regardless, themicrofluidic device 100 can be configured to be a nanofluidic device. Asillustrated in FIG. 1A, the microfluidic circuit 120 may include aplurality of microfluidic sequestration pens 124, 126, 128, and 130,where each sequestration pens may have one or more openings in fluidiccommunication with flow path 106. In some embodiments of the device ofFIG. 1A, the sequestration pens may have only a single opening influidic communication with the flow path 106. As discussed furtherbelow, the microfluidic sequestration pens comprise various features andstructures that have been optimized for retaining micro-objects in themicrofluidic device, such as microfluidic device 100, even when a medium180 is flowing through the flow path 106. Before turning to theforegoing, however, a brief description of microfluidic device 100 andsystem 150 is provided.

As generally illustrated in FIG. 1A, the microfluidic circuit 120 isdefined by an enclosure 102. Although the enclosure 102 can bephysically structured in different configurations, in the example shownin FIG. 1A the enclosure 102 is depicted as comprising a supportstructure 104 (e.g., a base), a microfluidic circuit structure 108, anda cover 110. The support structure 104, microfluidic circuit structure108, and cover 110 can be attached to each other. For example, themicrofluidic circuit structure 108 can be disposed on an inner surface109 of the support structure 104, and the cover 110 can be disposed overthe microfluidic circuit structure 108. Together with the supportstructure 104 and cover 110, the microfluidic circuit structure 108 candefine the elements of the microfluidic circuit 120.

The support structure 104 can be at the bottom and the cover 110 at thetop of the microfluidic circuit 120 as illustrated in FIG. 1A.Alternatively, the support structure 104 and the cover 110 can beconfigured in other orientations. For example, the support structure 104can be at the top and the cover 110 at the bottom of the microfluidiccircuit 120. Regardless, there can be one or more ports 107 eachcomprising a passage into or out of the enclosure 102. Examples of apassage include a valve, a gate, a pass-through hole, or the like. Asillustrated, port 107 is a pass-through hole created by a gap in themicrofluidic circuit structure 108. However, the port 107 can besituated in other components of the enclosure 102, such as the cover110. Only one port 107 is illustrated in FIG. 1A but the microfluidiccircuit 120 can have two or more ports 107. For example, there can be afirst port 107 that functions as an inlet for fluid entering themicrofluidic circuit 120, and there can be a second port 107 thatfunctions as an outlet for fluid exiting the microfluidic circuit 120.Whether a port 107 function as an inlet or an outlet can depend upon thedirection that fluid flows through flow path 106.

The support structure 104 can comprise one or more electrodes (notshown) and a substrate or a plurality of interconnected substrates. Forexample, the support structure 104 can comprise one or moresemiconductor substrates, each of which is electrically connected to anelectrode (e.g., all or a subset of the semiconductor substrates can beelectrically connected to a single electrode). The support structure 104can further comprise a printed circuit board assembly (“PCBA”). Forexample, the semiconductor substrate(s) can be mounted on a PCBA.

The microfluidic circuit structure 108 can define circuit elements ofthe microfluidic circuit 120. Such circuit elements can comprise spacesor regions that can be fluidly interconnected when microfluidic circuit120 is filled with fluid, such as flow regions (which may include or beone or more flow channels), chambers, pens, traps, and the like. In themicrofluidic circuit 120 illustrated in FIG. 1A, the microfluidiccircuit structure 108 comprises a frame 114 and a microfluidic circuitmaterial 116. The frame 114 can partially or completely enclose themicrofluidic circuit material 116. The frame 114 can be, for example, arelatively rigid structure substantially surrounding the microfluidiccircuit material 116. For example, the frame 114 can comprise a metalmaterial.

The microfluidic circuit material 116 can be patterned with cavities orthe like to define circuit elements and interconnections of themicrofluidic circuit 120. The microfluidic circuit material 116 cancomprise a flexible material, such as a flexible polymer (e.g. rubber,plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or thelike), which can be gas permeable. Other examples of materials that cancompose microfluidic circuit material 116 include molded glass, anetchable material such as silicone (e.g. photo-patternable silicone or“PPS”), photo-resist (e.g., SU8), or the like. In some embodiments, suchmaterials—and thus the microfluidic circuit material 116—can be rigidand/or substantially impermeable to gas. Regardless, microfluidiccircuit material 116 can be disposed on the support structure 104 andinside the frame 114.

The cover 110 can be an integral part of the frame 114 and/or themicrofluidic circuit material 116. Alternatively, the cover 110 can be astructurally distinct element, as illustrated in FIG. 1A. The cover 110can comprise the same or different materials than the frame 114 and/orthe microfluidic circuit material 116. Similarly, the support structure104 can be a separate structure from the frame 114 or microfluidiccircuit material 116 as illustrated, or an integral part of the frame114 or microfluidic circuit material 116. Likewise, the frame 114 andmicrofluidic circuit material 116 can be separate structures as shown inFIG. 1A or integral portions of the same structure.

In some embodiments, the cover 110 can comprise a rigid material. Therigid material may be glass or a material with similar properties. Insome embodiments, the cover 110 can comprise a deformable material. Thedeformable material can be a polymer, such as PDMS. In some embodiments,the cover 110 can comprise both rigid and deformable materials. Forexample, one or more portions of cover 110 (e.g., one or more portionspositioned over sequestration pens 124, 126, 128, 130) can comprise adeformable material that interfaces with rigid materials of the cover110. In some embodiments, the cover 110 can further include one or moreelectrodes. The one or more electrodes can comprise a conductive oxide,such as indium-tin-oxide (ITO), which may be coated on glass or asimilarly insulating material. Alternatively, the one or more electrodescan be flexible electrodes, such as single-walled nanotubes,multi-walled nanotubes, nanowires, clusters of electrically conductivenanoparticles, or combinations thereof, embedded in a deformablematerial, such as a polymer (e.g., PDMS). Flexible electrodes that canbe used in microfluidic devices have been described, for example, inU.S. 2012/0325665 (Chiou et al.), the contents of which are incorporatedherein by reference. In some embodiments, the cover 110 can be modified(e.g., by conditioning all or part of a surface that faces inward towardthe microfluidic circuit 120) to support cell adhesion, viability and/orgrowth. The modification may include a coating of a synthetic or naturalpolymer. In some embodiments, the cover 110 and/or the support structure104 can be transparent to light. The cover 110 may also include at leastone material that is gas permeable (e.g., PDMS or PPS).

FIG. 1A also shows a system 150 for operating and controllingmicrofluidic devices, such as microfluidic device 100. System 150includes an electrical power source 192, an imaging device (incorporatedwithin imaging module 164, and not explicitly illustrated in FIG. 1A),and a tilting device (part of tilting module 166, and not explicitlyillustrated in FIG. 1A).

The electrical power source 192 can provide electric power to themicrofluidic device 100 and/or tilting device 190, providing biasingvoltages or currents as needed. The electrical power source 192 can, forexample, comprise one or more alternating current (AC) and/or directcurrent (DC) voltage or current sources. The imaging device 194 (part ofimaging module 164, discussed below) can comprise a device, such as adigital camera, for capturing images inside microfluidic circuit 120. Insome instances, the imaging device 194 further comprises a detectorhaving a fast frame rate and/or high sensitivity (e.g. for low lightapplications). The imaging device 194 can also include a mechanism fordirecting stimulating radiation and/or light beams into the microfluidiccircuit 120 and collecting radiation and/or light beams reflected oremitted from the microfluidic circuit 120 (or micro-objects containedtherein). The emitted light beams may be in the visible spectrum andmay, e.g., include fluorescent emissions. The reflected light beams mayinclude reflected emissions originating from an LED or a wide spectrumlamp, such as a mercury lamp (e.g. a high pressure mercury lamp) or aXenon arc lamp. As discussed with respect to FIG. 3B, the imaging device194 may further include a microscope (or an optical train), which may ormay not include an eyepiece.

System 150 further comprises a tilting device 190 (part of tiltingmodule 166, discussed below) configured to rotate a microfluidic device100 about one or more axes of rotation. In some embodiments, the tiltingdevice 190 is configured to support and/or hold the enclosure 102comprising the microfluidic circuit 120 about at least one axis suchthat the microfluidic device 100 (and thus the microfluidic circuit 120)can be held in a level orientation (i.e. at 00 relative to x- andy-axes), a vertical orientation (i.e. at 900 relative to the x-axisand/or the y-axis), or any orientation therebetween. The orientation ofthe microfluidic device 100 (and the microfluidic circuit 120) relativeto an axis is referred to herein as the “tilt” of the microfluidicdevice 100 (and the microfluidic circuit 120). For example, the tiltingdevice 190 can tilt the microfluidic device 100 at 0.1°, 0.2°, 0.3°,0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°,25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relativeto the x-axis or any degree therebetween. The level orientation (andthus the x- and y-axes) is defined as normal to a vertical axis definedby the force of gravity. The tilting device can also tilt themicrofluidic device 100 (and the microfluidic circuit 120) to any degreegreater than 90° relative to the x-axis and/or y-axis, or tilt themicrofluidic device 100 (and the microfluidic circuit 120) 180° relativeto the x-axis or the y-axis in order to fully invert the microfluidicdevice 100 (and the microfluidic circuit 120). Similarly, in someembodiments, the tilting device 190 tilts the microfluidic device 100(and the microfluidic circuit 120) about an axis of rotation defined byflow path 106 or some other portion of microfluidic circuit 120.

In some instances, the microfluidic device 100 is tilted into a verticalorientation such that the flow path 106 is positioned above or below oneor more sequestration pens. The term “above” as used herein denotes thatthe flow path 106 is positioned higher than the one or moresequestration pens on a vertical axis defined by the force of gravity(i.e. an object in a sequestration pen above a flow path 106 would havea higher gravitational potential energy than an object in the flowpath). The term “below” as used herein denotes that the flow path 106 ispositioned lower than the one or more sequestration pens on a verticalaxis defined by the force of gravity (i.e. an object in a sequestrationpen below a flow path 106 would have a lower gravitational potentialenergy than an object in the flow path).

In some instances, the tilting device 190 tilts the microfluidic device100 about an axis that is parallel to the flow path 106. Moreover, themicrofluidic device 100 can be tilted to an angle of less than 90° suchthat the flow path 106 is located above or below one or moresequestration pens without being located directly above or below thesequestration pens. In other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis perpendicular to the flow path106. In still other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis that is neither parallel norperpendicular to the flow path 106.

System 150 can further include a media source 178. The media source 178(e.g., a container, reservoir, or the like) can comprise multiplesections or containers, each for holding a different fluidic medium 180.Thus, the media source 178 can be a device that is outside of andseparate from the microfluidic device 100, as illustrated in FIG. 1A.Alternatively, the media source 178 can be located in whole or in partinside the enclosure 102 of the microfluidic device 100. For example,the media source 178 can comprise reservoirs that are part of themicrofluidic device 100.

FIG. 1A also illustrates simplified block diagram depictions of examplesof control and monitoring equipment 152 that constitute part of system150 and can be utilized in conjunction with a microfluidic device 100.As shown, examples of such control and monitoring equipment 152 includea master controller 154 comprising a media module 160 for controllingthe media source 178, a motive module 162 for controlling movementand/or selection of micro-objects (not shown) and/or medium (e.g.,droplets of medium) in the microfluidic circuit 120, an imaging module164 for controlling an imaging device 194 (e.g., a camera, microscope,light source or any combination thereof) for capturing images (e.g.,digital images), and a tilting module 166 for controlling a tiltingdevice 190. The control equipment 152 can also include other modules 168for controlling, monitoring, or performing other functions with respectto the microfluidic device 100. As shown, the equipment 152 can furtherinclude a display device 170 and an input/output device 172.

The master controller 154 can comprise a control module 156 and adigital memory 158. The control module 156 can comprise, for example, adigital processor configured to operate in accordance with machineexecutable instructions (e.g., software, firmware, source code, or thelike) stored as non-transitory data or signals in the memory 158.Alternatively, or in addition, the control module 156 can comprisehardwired digital circuitry and/or analog circuitry. The media module160, motive module 162, imaging module 164, tilting module 166, and/orother modules 168 can be similarly configured. Thus, functions,processes acts, actions, or steps of a process discussed herein as beingperformed with respect to the microfluidic device 100 or any othermicrofluidic apparatus can be performed by any one or more of the mastercontroller 154, media module 160, motive module 162, imaging module 164,tilting module 166, and/or other modules 168 configured as discussedabove. Similarly, the master controller 154, media module 160, motivemodule 162, imaging module 164, tilting module 166, and/or other modules168 may be communicatively coupled to transmit and receive data used inany function, process, act, action or step discussed herein.

The media module 160 controls the media source 178. For example, themedia module 160 can control the media source 178 to input a selectedfluidic medium 180 into the enclosure 102 (e.g., through an inlet port107). The media module 160 can also control removal of media from theenclosure 102 (e.g., through an outlet port (not shown)). One or moremedia can thus be selectively input into and removed from themicrofluidic circuit 120. The media module 160 can also control the flowof fluidic medium 180 in the flow path 106 inside the microfluidiccircuit 120. For example, in some embodiments media module 160 stops theflow of media 180 in the flow path 106 and through the enclosure 102prior to the tilting module 166 causing the tilting device 190 to tiltthe microfluidic device 100 to a desired angle of incline.

The motive module 162 can be configured to control selection, trapping,and movement of micro-objects (not shown) in the microfluidic circuit120. As discussed below with respect to FIGS. 1B and 1C, the enclosure102 can comprise a dielectrophoresis (DEP), optoelectronic tweezers(OET) and/or opto-electrowetting (OEW) configuration (not shown in FIG.1A), and the motive module 162 can control the activation of electrodesand/or transistors (e.g., phototransistors) to select and movemicro-objects (not shown) and/or droplets of medium (not shown) in theflow path 106 and/or sequestration pens 124, 126, 128, 130.

The imaging module 164 can control the imaging device 194. For example,the imaging module 164 can receive and process image data from theimaging device 194. Image data from the imaging device 194 can compriseany type of information captured by the imaging device 194 (e.g., thepresence or absence of micro-objects, droplets of medium, accumulationof label, such as fluorescent label, etc.). Using the informationcaptured by the imaging device 194, the imaging module 164 can furthercalculate the position of objects (e.g., micro-objects, droplets ofmedium) and/or the rate of motion of such objects within themicrofluidic device 100.

The tilting module 166 can control the tilting motions of tilting device190. Alternatively, or in addition, the tilting module 166 can controlthe tilting rate and timing to optimize transfer of micro-objects to theone or more sequestration pens via gravitational forces. The tiltingmodule 166 is communicatively coupled with the imaging module 164 toreceive data describing the motion of micro-objects and/or droplets ofmedium in the microfluidic circuit 120. Using this data, the tiltingmodule 166 may adjust the tilt of the microfluidic circuit 120 in orderto adjust the rate at which micro-objects and/or droplets of medium movein the microfluidic circuit 120. The tilting module 166 may also usethis data to iteratively adjust the position of a micro-object and/ordroplet of medium in the microfluidic circuit 120.

In the example shown in FIG. 1A, the microfluidic circuit 120 isillustrated as comprising a microfluidic channel 122 and sequestrationpens 124, 126, 128, 130. Each pen comprises an opening to channel 122,but otherwise is enclosed such that the pens can substantially isolatemicro-objects inside the pen from fluidic medium 180 and/ormicro-objects in the flow path 106 of channel 122 or in other pens. Thewalls of the sequestration pen extend from the inner surface 109 of thebase to the inside surface of the cover 110 to provide enclosure. Theopening of the pen to the microfluidic channel 122 is oriented at anangle to the flow 106 of fluidic medium 180 such that flow 106 is notdirected into the pens. The flow may be tangential or orthogonal to theplane of the opening of the pen. In some instances, pens 124, 126, 128,130 are configured to physically corral one or more micro-objects withinthe microfluidic circuit 120. Sequestration pens in accordance with thepresent disclosure can comprise various shapes, surfaces and featuresthat are optimized for use with DEP, OET, OEW, fluid flow, and/orgravitational forces, as will be discussed and shown in detail below.

The microfluidic circuit 120 may comprise any number of microfluidicsequestration pens. Although five sequestration pens are shown,microfluidic circuit 120 may have fewer or more sequestration pens. Asshown, microfluidic sequestration pens 124, 126, 128, and 130 ofmicrofluidic circuit 120 each comprise differing features and shapeswhich may provide one or more benefits useful for maintaining,isolating, assaying or culturing biological micro-objects. In someembodiments, the microfluidic circuit 120 comprises a plurality ofidentical microfluidic sequestration pens.

In the embodiment illustrated in FIG. 1A, a single channel 122 and flowpath 106 is shown. However, other embodiments may contain multiplechannels 122, each configured to comprise a flow path 106. Themicrofluidic circuit 120 further comprises an inlet valve or port 107 influid communication with the flow path 106 and fluidic medium 180,whereby fluidic medium 180 can access channel 122 via the inlet port107. In some instances, the flow path 106 comprises a single path. Insome instances, the single path is arranged in a zigzag pattern wherebythe flow path 106 travels across the microfluidic device 100 two or moretimes in alternating directions.

In some instances, microfluidic circuit 120 comprises a plurality ofparallel channels 122 and flow paths 106, wherein the fluidic medium 180within each flow path 106 flows in the same direction. In someinstances, the fluidic medium within each flow path 106 flows in atleast one of a forward or reverse direction. In some instances, aplurality of sequestration pens is configured (e.g., relative to achannel 122) such that the sequestration pens can be loaded with targetmicro-objects in parallel.

In some embodiments, microfluidic circuit 120 further comprises one ormore micro-object traps 132. The traps 132 are generally formed in awall forming the boundary of a channel 122, and may be positionedopposite an opening of one or more of the microfluidic sequestrationpens 124, 126, 128, 130. In some embodiments, the traps 132 areconfigured to receive or capture a single micro-object from the flowpath 106. In some embodiments, the traps 132 are configured to receiveor capture a plurality of micro-objects from the flow path 106. In someinstances, the traps 132 comprise a volume approximately equal to thevolume of a single target micro-object.

The traps 132 may further comprise an opening which is configured toassist the flow of targeted micro-objects into the traps 132. In someinstances, the traps 132 comprise an opening having a height and widththat is approximately equal to the dimensions of a single targetmicro-object, whereby larger micro-objects are prevented from enteringinto the micro-object trap. The traps 132 may further comprise otherfeatures configured to assist in retention of targeted micro-objectswithin the trap 132. In some instances, the trap 132 is aligned with andsituated on the opposite side of a channel 122 relative to the openingof a microfluidic sequestration pen, such that upon tilting themicrofluidic device 100 about an axis parallel to the microfluidicchannel 122, the trapped micro-object exits the trap 132 at a trajectorythat causes the micro-object to fall into the opening of thesequestration pen. In some instances, the trap 132 comprises a sidepassage 134 that is smaller than the target micro-object in order tofacilitate flow through the trap 132 and thereby increase the likelihoodof capturing a micro-object in the trap 132.

In some embodiments, dielectrophoretic (DEP) forces are applied acrossthe fluidic medium 180 (e.g., in the flow path and/or in thesequestration pens) via one or more electrodes (not shown) tomanipulate, transport, separate and sort micro-objects located therein.For example, in some embodiments, DEP forces are applied to one or moreportions of microfluidic circuit 120 in order to transfer a singlemicro-object from the flow path 106 into a desired microfluidicsequestration pen. In some embodiments, DEP forces are used to prevent amicro-object within a sequestration pen (e.g., sequestration pen 124,126, 128, or 130) from being displaced therefrom. Further, in someembodiments, DEP forces are used to selectively remove a micro-objectfrom a sequestration pen that was previously collected in accordancewith the embodiments of the current disclosure. In some embodiments, theDEP forces comprise optoelectronic tweezer (OET) forces.

In other embodiments, optoelectrowetting (OEW) forces are applied to oneor more positions in the support structure 104 (and/or the cover 110) ofthe microfluidic device 100 (e.g., positions helping to define the flowpath and/or the sequestration pens) via one or more electrodes (notshown) to manipulate, transport, separate and sort droplets located inthe microfluidic circuit 120. For example, in some embodiments, OEWforces are applied to one or more positions in the support structure 104(and/or the cover 110) in order to transfer a single droplet from theflow path 106 into a desired microfluidic sequestration pen. In someembodiments, OEW forces are used to prevent a droplet within asequestration pen (e.g., sequestration pen 124, 126, 128, or 130) frombeing displaced therefrom. Further, in some embodiments, OEW forces areused to selectively remove a droplet from a sequestration pen that waspreviously collected in accordance with the embodiments of the currentdisclosure.

In some embodiments, DEP and/or OEW forces are combined with otherforces, such as flow and/or gravitational force, so as to manipulate,transport, separate and sort micro-objects and/or droplets within themicrofluidic circuit 120. For example, the enclosure 102 can be tilted(e.g., by tilting device 190) to position the flow path 106 andmicro-objects located therein above the microfluidic sequestration pens,and the force of gravity can transport the micro-objects and/or dropletsinto the pens. In some embodiments, the DEP and/or OEW forces can beapplied prior to the other forces. In other embodiments, the DEP and/orOEW forces can be applied after the other forces. In still otherinstances, the DEP and/or OEW forces can be applied at the same time asthe other forces or in an alternating manner with the other forces.

FIGS. 1B, 1C, and 2A-2H illustrates various embodiments of microfluidicdevices that can be used in the practice of the embodiments of thepresent disclosure. FIG. 1B depicts an embodiment in which themicrofluidic device 200 is configured as an optically-actuatedelectrokinetic device. A variety of optically-actuated electrokineticdevices are known in the art, including devices having an optoelectronictweezer (OET) configuration and devices having an opto-electrowetting(OEW) configuration. Examples of suitable OET configurations areillustrated in the following U.S. patent documents, each of which isincorporated herein by reference in its entirety: U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355); andU.S. Pat. No. 7,956,339 (Ohta et al.). Examples of OEW configurationsare illustrated in U.S. Pat. No. 6,958,132 (Chiou et al.) and U.S.Patent Application Publication No. 2012/0024708 (Chiou et al.), both ofwhich are incorporated by reference herein in their entirety. Yetanother example of an optically-actuated electrokinetic device includesa combined OET/OEW configuration, examples of which are shown in U.S.Patent Publication Nos. 20150306598 (Khandros et al.) and 20150306599(Khandros et al.) and their corresponding PCT Publications WO2015/164846and WO2015/164847, all of which are incorporated herein by reference intheir entirety.

Examples of microfluidic devices having pens in which biologicalmicro-objects can be placed, cultured, and/or monitored have beendescribed, for example, in US 2014/0116881 (application Ser. No.14/060,117, filed Oct. 22, 2013), US 2015/0151298 (application Ser. No.14/520,568, filed Oct. 22, 2014), and US 2015/0165436 (application Ser.No. 14/521,447, filed Oct. 22, 2014), each of which is incorporatedherein by reference in its entirety. U.S. application Ser. Nos.14/520,568 and 14/521,447 also describe exemplary methods of analyzingsecretions of cells cultured in a microfluidic device. Each of theforegoing applications further describes microfluidic devices configuredto produce dielectrophoretic (DEP) forces, such as optoelectronictweezers (OET) or configured to provide opto-electro wetting (OEW). Forexample, the optoelectronic tweezers device illustrated in FIG. 2 of US2014/0116881 is an example of a device that can be utilized inembodiments of the present disclosure to select and move an individualbiological micro-object or a group of biological micro-objects.

Microfluidic Device Motive Configurations.

As described above, the control and monitoring equipment of the systemcan comprise a motive module for selecting and moving objects, such asmicro-objects or droplets, in the microfluidic circuit of a microfluidicdevice. The microfluidic device can have a variety of motiveconfigurations, depending upon the type of object being moved and otherconsiderations. For example, a dielectrophoresis (DEP) configuration canbe utilized to select and move micro-objects in the microfluidiccircuit. Thus, the support structure 104 and/or cover 110 of themicrofluidic device 100 can comprise a DEP configuration for selectivelyinducing DEP forces on micro-objects in a fluidic medium 180 in themicrofluidic circuit 120 and thereby select, capture, and/or moveindividual micro-objects or groups of micro-objects. Alternatively, thesupport structure 104 and/or cover 110 of the microfluidic device 100can comprise an electrowetting (EW) configuration for selectivelyinducing EW forces on droplets in a fluidic medium 180 in themicrofluidic circuit 120 and thereby select, capture, and/or moveindividual droplets or groups of droplets.

One example of a microfluidic device 200 comprising a DEP configurationis illustrated in FIGS. 1B and 1C. While for purposes of simplicityFIGS. 1B and 1C show a side cross-sectional view and a topcross-sectional view, respectively, of a portion of an enclosure 102 ofthe microfluidic device 200 having a region/chamber 202, it should beunderstood that the region/chamber 202 may be part of a fluidic circuitelement having a more detailed structure, such as a growth chamber, asequestration pen, a flow region, or a flow channel. Furthermore, themicrofluidic device 200 may include other fluidic circuit elements. Forexample, the microfluidic device 200 can include a plurality of growthchambers or sequestration pens and/or one or more flow regions or flowchannels, such as those described herein with respect to microfluidicdevice 100. A DEP configuration may be incorporated into any suchfluidic circuit elements of the microfluidic device 200, or selectportions thereof. It should be further appreciated that any of the aboveor below described microfluidic device components and system componentsmay be incorporated in and/or used in combination with the microfluidicdevice 200. For example, system 150 including control and monitoringequipment 152, described above, may be used with microfluidic device200, including one or more of the media module 160, motive module 162,imaging module 164, tilting module 166, and other modules 168.

As seen in FIG. 1B, the microfluidic device 200 includes a supportstructure 104 having a bottom electrode 204 and an electrode activationsubstrate 206 overlying the bottom electrode 204, and a cover 110 havinga top electrode 210, with the top electrode 210 spaced apart from thebottom electrode 204. The top electrode 210 and the electrode activationsubstrate 206 define opposing surfaces of the region/chamber 202. Amedium 180 contained in the region/chamber 202 thus provides a resistiveconnection between the top electrode 210 and the electrode activationsubstrate 206. A power source 212 configured to be connected to thebottom electrode 204 and the top electrode 210 and create a biasingvoltage between the electrodes, as required for the generation of DEPforces in the region/chamber 202, is also shown. The power source 212can be, for example, an alternating current (AC) power source.

In certain embodiments, the microfluidic device 200 illustrated in FIGS.1B and 1C can have an optically-actuated DEP configuration. Accordingly,changing patterns of light 218 from the light source 216, which may becontrolled by the motive module 162, can selectively activate anddeactivate changing patterns of DEP electrodes at regions 214 of theinner surface 208 of the electrode activation substrate 206.(Hereinafter the regions 214 of a microfluidic device having a DEPconfiguration are referred to as “DEP electrode regions.”) Asillustrated in FIG. 1C, a light pattern 218 directed onto the innersurface 208 of the electrode activation substrate 206 can illuminateselect DEP electrode regions 214 a (shown in white) in a pattern, suchas a square. The non-illuminated DEP electrode regions 214(cross-hatched) are hereinafter referred to as “dark” DEP electroderegions 214. The relative electrical impedance through the DEP electrodeactivation substrate 206 (i.e., from the bottom electrode 204 up to theinner surface 208 of the electrode activation substrate 206 whichinterfaces with the medium 180 in the flow region 106) is greater thanthe relative electrical impedance through the medium 180 in theregion/chamber 202 (i.e., from the inner surface 208 of the electrodeactivation substrate 206 to the top electrode 210 of the cover 110) ateach dark DEP electrode region 214. An illuminated DEP electrode region214 a, however, exhibits a reduced relative impedance through theelectrode activation substrate 206 that is less than the relativeimpedance through the medium 180 in the region/chamber 202 at eachilluminated DEP electrode region 214 a.

With the power source 212 activated, the foregoing DEP configurationcreates an electric field gradient in the fluidic medium 180 betweenilluminated DEP electrode regions 214 a and adjacent dark DEP electroderegions 214, which in turn creates local DEP forces that attract orrepel nearby micro-objects (not shown) in the fluidic medium 180. DEPelectrodes that attract or repel micro-objects in the fluidic medium 180can thus be selectively activated and deactivated at many different suchDEP electrode regions 214 at the inner surface 208 of the region/chamber202 by changing light patterns 218 projected from a light source 216into the microfluidic device 200. Whether the DEP forces attract orrepel nearby micro-objects can depend on such parameters as thefrequency of the power source 212 and the dielectric properties of themedium 180 and/or micro-objects (not shown).

The square pattern 220 of illuminated DEP electrode regions 214 aillustrated in FIG. 1C is an example only. Any pattern of the DEPelectrode regions 214 can be illuminated (and thereby activated) by thepattern of light 218 projected into the microfluidic device 200, and thepattern of illuminated/activated DEP electrode regions 214 can berepeatedly changed by changing or moving the light pattern 218.

In some embodiments, the electrode activation substrate 206 can compriseor consist of a photoconductive material. In such embodiments, the innersurface 208 of the electrode activation substrate 206 can befeatureless. For example, the electrode activation substrate 206 cancomprise or consist of a layer of hydrogenated amorphous silicon(a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen(calculated as 100*the number of hydrogen atoms/the total number ofhydrogen and silicon atoms). The layer of a-Si:H can have a thickness ofabout 500 nm to about 2.0 μm. In such embodiments, the DEP electroderegions 214 can be created anywhere and in any pattern on the innersurface 208 of the electrode activation substrate 206, in accordancewith the light pattern 218. The number and pattern of the DEP electroderegions 214 thus need not be fixed, but can correspond to the lightpattern 218. Examples of microfluidic devices having a DEP configurationcomprising a photoconductive layer such as discussed above have beendescribed, for example, in U.S. Pat. No. RE 44,711 (Wu et al.)(originally issued as U.S. Pat. No. 7,612,355), the entire contents ofwhich are incorporated herein by reference.

In other embodiments, the electrode activation substrate 206 cancomprise a substrate comprising a plurality of doped layers,electrically insulating layers (or regions), and electrically conductivelayers that form semiconductor integrated circuits, such as is known insemiconductor fields. For example, the electrode activation substrate206 can comprise a plurality of phototransistors, including, forexample, lateral bipolar phototransistors, each phototransistorcorresponding to a DEP electrode region 214. Alternatively, theelectrode activation substrate 206 can comprise electrodes (e.g.,conductive metal electrodes) controlled by phototransistor switches,with each such electrode corresponding to a DEP electrode region 214.The electrode activation substrate 206 can include a pattern of suchphototransistors or phototransistor-controlled electrodes. The pattern,for example, can be an array of substantially square phototransistors orphototransistor-controlled electrodes arranged in rows and columns, suchas shown in FIG. 2B. Alternatively, the pattern can be an array ofsubstantially hexagonal phototransistors or phototransistor-controlledelectrodes that form a hexagonal lattice. Regardless of the pattern,electric circuit elements can form electrical connections between theDEP electrode regions 214 at the inner surface 208 of the electrodeactivation substrate 206 and the bottom electrode 210, and thoseelectrical connections (i.e., phototransistors or electrodes) can beselectively activated and deactivated by the light pattern 218. When notactivated, each electrical connection can have high impedance such thatthe relative impedance through the electrode activation substrate 206(i.e., from the bottom electrode 204 to the inner surface 208 of theelectrode activation substrate 206 which interfaces with the medium 180in the region/chamber 202) is greater than the relative impedancethrough the medium 180 (i.e., from the inner surface 208 of theelectrode activation substrate 206 to the top electrode 210 of the cover110) at the corresponding DEP electrode region 214. When activated bylight in the light pattern 218, however, the relative impedance throughthe electrode activation substrate 206 is less than the relativeimpedance through the medium 180 at each illuminated DEP electroderegion 214, thereby activating the DEP electrode at the correspondingDEP electrode region 214 as discussed above. DEP electrodes that attractor repel micro-objects (not shown) in the medium 180 can thus beselectively activated and deactivated at many different DEP electroderegions 214 at the inner surface 208 of the electrode activationsubstrate 206 in the region/chamber 202 in a manner determined by thelight pattern 218.

Examples of microfluidic devices having electrode activation substratesthat comprise phototransistors have been described, for example, in U.S.Pat. No. 7,956,339 (Ohta et al.) (see, e.g., device 300 illustrated inFIGS. 21 and 22, and descriptions thereof), the entire contents of whichare incorporated herein by reference. Examples of microfluidic deviceshaving electrode activation substrates that comprise electrodescontrolled by phototransistor switches have been described, for example,in U.S. Patent Publication No. 2014/0124370 (Short et al.) (see, e.g.,devices 200, 400, 500, 600, and 900 illustrated throughout the drawings,and descriptions thereof), the entire contents of which are incorporatedherein by reference.

In some embodiments of a DEP configured microfluidic device, the topelectrode 210 is part of a first wall (or cover 110) of the enclosure102, and the electrode activation substrate 206 and bottom electrode 204are part of a second wall (or support structure 104) of the enclosure102. The region/chamber 202 can be between the first wall and the secondwall. In other embodiments, the electrode 210 is part of the second wall(or support structure 104) and one or both of the electrode activationsubstrate 206 and/or the electrode 210 are part of the first wall (orcover 110). Moreover, the light source 216 can alternatively be used toilluminate the enclosure 102 from below.

With the microfluidic device 200 of FIGS. 1B-1C having a DEPconfiguration, the motive module 162 can select a micro-object (notshown) in the medium 180 in the region/chamber 202 by projecting a lightpattern 218 into the microfluidic device 200 to activate a first set ofone or more DEP electrodes at DEP electrode regions 214 a of the innersurface 208 of the electrode activation substrate 206 in a pattern(e.g., square pattern 220) that surrounds and captures the micro-object.The motive module 162 can then move the in situ-generated capturedmicro-object by moving the light pattern 218 relative to themicrofluidic device 200 to activate a second set of one or more DEPelectrodes at DEP electrode regions 214. Alternatively, the microfluidicdevice 200 can be moved relative to the light pattern 218.

In other embodiments, the microfluidic device 200 can have a DEPconfiguration that does not rely upon light activation of DEP electrodesat the inner surface 208 of the electrode activation substrate 206. Forexample, the electrode activation substrate 206 can comprise selectivelyaddressable and energizable electrodes positioned opposite to a surfaceincluding at least one electrode (e.g., cover 110). Switches (e.g.,transistor switches in a semiconductor substrate) may be selectivelyopened and closed to activate or inactivate DEP electrodes at DEPelectrode regions 214, thereby creating a net DEP force on amicro-object (not shown) in region/chamber 202 in the vicinity of theactivated DEP electrodes. Depending on such characteristics as thefrequency of the power source 212 and the dielectric properties of themedium (not shown) and/or micro-objects in the region/chamber 202, theDEP force can attract or repel a nearby micro-object. By selectivelyactivating and deactivating a set of DEP electrodes (e.g., at a set ofDEP electrodes regions 214 that forms a square pattern 220), one or moremicro-objects in region/chamber 202 can be trapped and moved within theregion/chamber 202. The motive module 162 in FIG. 1A can control suchswitches and thus activate and deactivate individual ones of the DEPelectrodes to select, trap, and move particular micro-objects (notshown) around the region/chamber 202. Microfluidic devices having a DEPconfiguration that includes selectively addressable and energizableelectrodes are known in the art and have been described, for example, inU.S. Pat. No. 6,294,063 (Becker et al.) and U.S. Pat. No. 6,942,776(Medoro), the entire contents of which are incorporated herein byreference.

As yet another example, the microfluidic device 200 can have anelectrowetting (EW) configuration, which can be in place of the DEPconfiguration or can be located in a portion of the microfluidic device200 that is separate from the portion which has the DEP configuration.The EW configuration can be an opto-electrowetting configuration or anelectrowetting on dielectric (EWOD) configuration, both of which areknown in the art. In some EW configurations, the support structure 104has an electrode activation substrate 206 sandwiched between adielectric layer (not shown) and the bottom electrode 204. Thedielectric layer can comprise a hydrophobic material and/or can becoated with a hydrophobic material, as described below. For microfluidicdevices 200 that have an EW configuration, the inner surface 208 of thesupport structure 104 is the inner surface of the dielectric layer orits hydrophobic coating.

The dielectric layer (not shown) can comprise one or more oxide layers,and can have a thickness of about 50 nm to about 250 nm (e.g., about 125nm to about 175 nm). In certain embodiments, the dielectric layer maycomprise a layer of oxide, such as a metal oxide (e.g., aluminum oxideor hafnium oxide). In certain embodiments, the dielectric layer cancomprise a dielectric material other than a metal oxide, such as siliconoxide or a nitride. Regardless of the exact composition and thickness,the dielectric layer can have an impedance of about 10 kOhms to about 50kOhms.

In some embodiments, the surface of the dielectric layer that facesinward toward region/chamber 202 is coated with a hydrophobic material.The hydrophobic material can comprise, for example, fluorinated carbonmolecules. Examples of fluorinated carbon molecules includeperfluoro-polymers such as polytetrafluoroethylene (e.g., TEFLON®) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (e.g., CYTOP™).Molecules that make up the hydrophobic material can be covalently bondedto the surface of the dielectric layer. For example, molecules of thehydrophobic material can be covalently bound to the surface of thedielectric layer by means of a linker such as a siloxane group, aphosphonic acid group, or a thiol group. Thus, in some embodiments, thehydrophobic material can comprise alkyl-terminated siloxane,alkyl-termination phosphonic acid, or alkyl-terminated thiol. The alkylgroup can be long-chain hydrocarbons (e.g., having a chain of at least10 carbons, or at least 16, 18, 20, 22, or more carbons). Alternatively,fluorinated (or perfluorinated) carbon chains can be used in place ofthe alkyl groups. Thus, for example, the hydrophobic material cancomprise fluoroalkyl-terminated siloxane, fluoroalkyl-terminatedphosphonic acid, or fluoroalkyl-terminated thiol. In some embodiments,the hydrophobic coating has a thickness of about 10 nm to about 50 nm.In other embodiments, the hydrophobic coating has a thickness of lessthan 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm).

In some embodiments, the cover 110 of a microfluidic device 200 havingan electrowetting configuration is coated with a hydrophobic material(not shown) as well. The hydrophobic material can be the samehydrophobic material used to coat the dielectric layer of the supportstructure 104, and the hydrophobic coating can have a thickness that issubstantially the same as the thickness of the hydrophobic coating onthe dielectric layer of the support structure 104. Moreover, the cover110 can comprise an electrode activation substrate 206 sandwichedbetween a dielectric layer and the top electrode 210, in the manner ofthe support structure 104. The electrode activation substrate 206 andthe dielectric layer of the cover 110 can have the same compositionand/or dimensions as the electrode activation substrate 206 and thedielectric layer of the support structure 104. Thus, the microfluidicdevice 200 can have two electrowetting surfaces.

In some embodiments, the electrode activation substrate 206 can comprisea photoconductive material, such as described above. Accordingly, incertain embodiments, the electrode activation substrate 206 can compriseor consist of a layer of hydrogenated amorphous silicon (a-Si:H). Thea-Si:H can comprise, for example, about 8% to 40% hydrogen (calculatedas 100*the number of hydrogen atoms/the total number of hydrogen andsilicon atoms). The layer of a-Si:H can have a thickness of about 500 nmto about 2.0 μm. Alternatively, the electrode activation substrate 206can comprise electrodes (e.g., conductive metal electrodes) controlledby phototransistor switches, as described above. Microfluidic deviceshaving an opto-electrowetting configuration are known in the art and/orcan be constructed with electrode activation substrates known in theart. For example, U.S. Pat. No. 6,958,132 (Chiou et al.), the entirecontents of which are incorporated herein by reference, disclosesopto-electrowetting configurations having a photoconductive materialsuch as a-Si:H, while U.S. Patent Publication No. 2014/0124370 (Short etal.), referenced above, discloses electrode activation substrates havingelectrodes controlled by phototransistor switches.

The microfluidic device 200 thus can have an opto-electrowettingconfiguration, and light patterns 218 can be used to activatephotoconductive EW regions or photoresponsive EW electrodes in theelectrode activation substrate 206. Such activated EW regions or EWelectrodes of the electrode activation substrate 206 can generate anelectrowetting force at the inner surface 208 of the support structure104 (i.e., the inner surface of the overlaying dielectric layer or itshydrophobic coating). By changing the light patterns 218 (or movingmicrofluidic device 200 relative to the light source 216) incident onthe electrode activation substrate 206, droplets (e.g., containing anaqueous medium, solution, or solvent) contacting the inner surface 208of the support structure 104 can be moved through an immiscible fluid(e.g., an oil medium) present in the region/chamber 202.

In other embodiments, microfluidic devices 200 can have an EWODconfiguration, and the electrode activation substrate 206 can compriseselectively addressable and energizable electrodes that do not rely uponlight for activation. The electrode activation substrate 206 thus caninclude a pattern of such electrowetting (EW) electrodes. The pattern,for example, can be an array of substantially square EW electrodesarranged in rows and columns, such as shown in FIG. 2B. Alternatively,the pattern can be an array of substantially hexagonal EW electrodesthat form a hexagonal lattice. Regardless of the pattern, the EWelectrodes can be selectively activated (or deactivated) by electricalswitches (e.g., transistor switches in a semiconductor substrate). Byselectively activating and deactivating EW electrodes in the electrodeactivation substrate 206, droplets (not shown) contacting the innersurface 208 of the overlaying dielectric layer or its hydrophobiccoating can be moved within the region/chamber 202. The motive module162 in FIG. 1A can control such switches and thus activate anddeactivate individual EW electrodes to select and move particulardroplets around region/chamber 202. Microfluidic devices having a EWODconfiguration with selectively addressable and energizable electrodesare known in the art and have been described, for example, in U.S. Pat.No. 8,685,344 (Sundarsan et al.), the entire contents of which areincorporated herein by reference.

Regardless of the configuration of the microfluidic device 200, a powersource 212 can be used to provide a potential (e.g., an AC voltagepotential) that powers the electrical circuits of the microfluidicdevice 200. The power source 212 can be the same as, or a component of,the power source 192 referenced in FIG. 1. Power source 212 can beconfigured to provide an AC voltage and/or current to the top electrode210 and the bottom electrode 204. For an AC voltage, the power source212 can provide a frequency range and an average or peak power (e.g.,voltage or current) range sufficient to generate net DEP forces (orelectrowetting forces) strong enough to trap and move individualmicro-objects (not shown) in the region/chamber 202, as discussed above,and/or to change the wetting properties of the inner surface 208 of thesupport structure 104 (i.e., the dielectric layer and/or the hydrophobiccoating on the dielectric layer) in the region/chamber 202, as alsodiscussed above. Such frequency ranges and average or peak power rangesare known in the art. See, e.g., U.S. Pat. No. 6,958,132 (Chiou et al.),U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No.7,612,355), and US Patent Application Publication Nos. US2014/0124370(Short et al.), US2015/0306598 (Khandros et al.), and US2015/0306599(Khandros et al.).

Sequestration Pens.

Non-limiting examples of generic sequestration pens 224, 226, and 228are shown within the microfluidic device 230 depicted in FIGS. 2A-2C.Each sequestration pen 224, 226, and 228 can comprise an isolationstructure 232 defining an isolation region 240 and a connection region236 fluidically connecting the isolation region 240 to a channel 122.The connection region 236 can comprise a proximal opening 234 to themicrofluidic channel 122 and a distal opening 238 to the isolationregion 240. The connection region 236 can be configured so that themaximum penetration depth of a flow of a fluidic medium (not shown)flowing from the microfluidic channel 122 into the sequestration pen224, 226, 228 does not extend into the isolation region 240. Thus, dueto the connection region 236, a micro-object (not shown) or othermaterial (not shown) disposed in an isolation region 240 of asequestration pen 224, 226, 228 can thus be isolated from, and notsubstantially affected by, a flow of medium 180 in the microfluidicchannel 122.

The sequestration pens 224, 226, and 228 of FIGS. 2A-2C each have asingle opening which opens directly to the microfluidic channel 122. Theopening of the sequestration pen opens laterally from the microfluidicchannel 122. The electrode activation substrate 206 underlays both themicrofluidic channel 122 and the sequestration pens 224, 226, and 228.The upper surface of the electrode activation substrate 206 within theenclosure of a sequestration pen, forming the floor of the sequestrationpen, is disposed at the same level or substantially the same level ofthe upper surface the of electrode activation substrate 206 within themicrofluidic channel 122 (or flow region if a channel is not present),forming the floor of the flow channel (or flow region, respectively) ofthe microfluidic device. The electrode activation substrate 206 may befeatureless or may have an irregular or patterned surface that variesfrom its highest elevation to its lowest depression by less than about 3microns, 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5microns, 0.4 microns, 0.2 microns, 0.1 microns or less. The variation ofelevation in the upper surface of the substrate across both themicrofluidic channel 122 (or flow region) and sequestration pens may beless than about 3%, 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the heightof the walls of the sequestration pen or walls of the microfluidicdevice. While described in detail for the microfluidic device 200, thisalso applies to any of the microfluidic devices 100, 230, 250, 280, 290described herein.

The microfluidic channel 122 can thus be an example of a swept region,and the isolation regions 240 of the sequestration pens 224, 226, 228can be examples of unswept regions. As noted, the microfluidic channel122 and sequestration pens 224, 226, 228 can be configured to containone or more fluidic media 180. In the example shown in FIGS. 2A-2B, theports 222 are connected to the microfluidic channel 122 and allow afluidic medium 180 to be introduced into or removed from themicrofluidic device 230. Prior to introduction of the fluidic medium180, the microfluidic device may be primed with a gas such as carbondioxide gas. Once the microfluidic device 230 contains the fluidicmedium 180, the flow 242 of fluidic medium 180 in the microfluidicchannel 122 can be selectively generated and stopped. For example, asshown, the ports 222 can be disposed at different locations (e.g.,opposite ends) of the microfluidic channel 122, and a flow 242 of mediumcan be created from one port 222 functioning as an inlet to another port222 functioning as an outlet.

FIG. 2C illustrates a detailed view of an example of a sequestration pen224 according to the present disclosure. Examples of micro-objects 246are also shown.

As is known, a flow 242 of fluidic medium 180 in a microfluidic channel122 past a proximal opening 234 of sequestration pen 224 can cause asecondary flow 244 of the medium 180 into and/or out of thesequestration pen 224. To isolate micro-objects 246 in the isolationregion 240 of a sequestration pen 224 from the secondary flow 244, thelength L_(con) of the connection region 236 of the sequestration pen 224(i.e., from the proximal opening 234 to the distal opening 238) shouldbe greater than the penetration depth D_(p) of the secondary flow 244into the connection region 236. The penetration depth D_(p) of thesecondary flow 244 depends upon the velocity of the fluidic medium 180flowing in the microfluidic channel 122 and various parameters relatingto the configuration of the microfluidic channel 122 and the proximalopening 234 of the connection region 236 to the microfluidic channel122. For a given microfluidic device, the configurations of themicrofluidic channel 122 and the opening 234 will be fixed, whereas therate of flow 242 of fluidic medium 180 in the microfluidic channel 122will be variable. Accordingly, for each sequestration pen 224, a maximalvelocity V_(max) for the flow 242 of fluidic medium 180 in channel 122can be identified that ensures that the penetration depth D_(p) of thesecondary flow 244 does not exceed the length L_(con) of the connectionregion 236. As long as the rate of the flow 242 of fluidic medium 180 inthe microfluidic channel 122 does not exceed the maximum velocityV_(max), the resulting secondary flow 244 can be limited to themicrofluidic channel 122 and the connection region 236 and kept out ofthe isolation region 240. The flow 242 of medium 180 in the microfluidicchannel 122 will thus not draw micro-objects 246 out of the isolationregion 240. Rather, micro-objects 246 located in the isolation region240 will stay in the isolation region 240 regardless of the flow 242 offluidic medium 180 in the microfluidic channel 122.

Moreover, as long as the rate of flow 242 of medium 180 in themicrofluidic channel 122 does not exceed V_(max), the flow 242 offluidic medium 180 in the microfluidic channel 122 will not movemiscellaneous particles (e.g., microparticles and/or nanoparticles) fromthe microfluidic channel 122 into the isolation region 240 of asequestration pen 224. Having the length L_(con) of the connectionregion 236 be greater than the maximum penetration depth D_(p) of thesecondary flow 244 can thus prevent contamination of one sequestrationpen 224 with miscellaneous particles from the microfluidic channel 122or another sequestration pen (e.g., sequestration pens 226, 228 in FIG.2D).

Because the microfluidic channel 122 and the connection regions 236 ofthe sequestration pens 224, 226, 228 can be affected by the flow 242 ofmedium 180 in the microfluidic channel 122, the microfluidic channel 122and connection regions 236 can be deemed swept (or flow) regions of themicrofluidic device 230. The isolation regions 240 of the sequestrationpens 224, 226, 228, on the other hand, can be deemed unswept (ornon-flow) regions. For example, components (not shown) in a firstfluidic medium 180 in the microfluidic channel 122 can mix with a secondfluidic medium 248 in the isolation region 240 substantially only bydiffusion of components of the first medium 180 from the microfluidicchannel 122 through the connection region 236 and into the secondfluidic medium 248 in the isolation region 240. Similarly, components(not shown) of the second medium 248 in the isolation region 240 can mixwith the first medium 180 in the microfluidic channel 122 substantiallyonly by diffusion of components of the second medium 248 from theisolation region 240 through the connection region 236 and into thefirst medium 180 in the microfluidic channel 122. In some embodiments,the extent of fluidic medium exchange between the isolation region of asequestration pen and the flow region by diffusion is greater than about90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about 99% offluidic exchange. The first medium 180 can be the same medium or adifferent medium than the second medium 248. Moreover, the first medium180 and the second medium 248 can start out being the same, then becomedifferent (e.g., through conditioning of the second medium 248 by one ormore cells in the isolation region 240, or by changing the medium 180flowing through the microfluidic channel 122).

The maximum penetration depth D_(p) of the secondary flow 244 caused bythe flow 242 of fluidic medium 180 in the microfluidic channel 122 candepend on a number of parameters, as mentioned above. Examples of suchparameters include: the shape of the microfluidic channel 122 (e.g., themicrofluidic channel can direct medium into the connection region 236,divert medium away from the connection region 236, or direct medium in adirection substantially perpendicular to the proximal opening 234 of theconnection region 236 to the microfluidic channel 122); a width W_(ch)(or cross-sectional area) of the microfluidic channel 122 at theproximal opening 234; and a width W_(con) (or cross-sectional area) ofthe connection region 236 at the proximal opening 234; the velocity V ofthe flow 242 of fluidic medium 180 in the microfluidic channel 122; theviscosity of the first medium 180 and/or the second medium 248, or thelike.

In some embodiments, the dimensions of the microfluidic channel 122 andsequestration pens 224, 226, 228 can be oriented as follows with respectto the vector of the flow 242 of fluidic medium 180 in the microfluidicchannel 122: the microfluidic channel width W_(ch) (or cross-sectionalarea of the microfluidic channel 122) can be substantially perpendicularto the flow 242 of medium 180; the width W_(con) (or cross-sectionalarea) of the connection region 236 at opening 234 can be substantiallyparallel to the flow 242 of medium 180 in the microfluidic channel 122;and/or the length L_(con) of the connection region can be substantiallyperpendicular to the flow 242 of medium 180 in the microfluidic channel122. The foregoing are examples only, and the relative position of themicrofluidic channel 122 and sequestration pens 224, 226, 228 can be inother orientations with respect to each other.

As illustrated in FIG. 2C, the width W_(con) of the connection region236 can be uniform from the proximal opening 234 to the distal opening238. The width W_(con) of the connection region 236 at the distalopening 238 can thus be any of the values identified herein for thewidth W_(con) of the connection region 236 at the proximal opening 234.Alternatively, the width W_(con) of the connection region 236 at thedistal opening 238 can be larger than the width W_(con) of theconnection region 236 at the proximal opening 234.

As illustrated in FIG. 2C, the width of the isolation region 240 at thedistal opening 238 can be substantially the same as the width W_(con) ofthe connection region 236 at the proximal opening 234. The width of theisolation region 240 at the distal opening 238 can thus be any of thevalues identified herein for the width W_(con) of the connection region236 at the proximal opening 234. Alternatively, the width of theisolation region 240 at the distal opening 238 can be larger or smallerthan the width W_(con) of the connection region 236 at the proximalopening 234. Moreover, the distal opening 238 may be smaller than theproximal opening 234 and the width W_(con) of the connection region 236may be narrowed between the proximal opening 234 and distal opening 238.For example, the connection region 236 may be narrowed between theproximal opening and the distal opening, using a variety of differentgeometries (e.g. chamfering the connection region, beveling theconnection region). Further, any part or subpart of the connectionregion 236 may be narrowed (e.g. a portion of the connection regionadjacent to the proximal opening 234).

FIGS. 2D-2F depict another exemplary embodiment of a microfluidic device250 containing a microfluidic circuit 262 and flow channels 264, whichare variations of the respective microfluidic device 100, circuit 132and channel 134 of FIG. 1A. The microfluidic device 250 also has aplurality of sequestration pens 266 that are additional variations ofthe above-described sequestration pens 124, 126, 128, 130, 224, 226 or228. In particular, it should be appreciated that the sequestration pens266 of device 250 shown in FIGS. 2D-2F can replace any of theabove-described sequestration pens 124, 126, 128, 130, 224, 226 or 228in devices 100, 200, 230, 280, 290. Likewise, the microfluidic device250 is another variant of the microfluidic device 100, and may also havethe same or a different DEP configuration as the above-describedmicrofluidic device 100, 200, 230, 280, 290, as well as any of the othermicrofluidic system components described herein.

The microfluidic device 250 of FIGS. 2D-2F comprises a support structure(not visible in FIGS. 2D-2F, but can be the same or generally similar tothe support structure 104 of device 100 depicted in FIG. 1A), amicrofluidic circuit structure 256, and a cover (not visible in FIGS.2D-2F, but can be the same or generally similar to the cover 122 ofdevice 100 depicted in FIG. 1A). The microfluidic circuit structure 256includes a frame 252 and microfluidic circuit material 260, which can bethe same as or generally similar to the frame 114 and microfluidiccircuit material 116 of device 100 shown in FIG. 1A. As shown in FIG.2D, the microfluidic circuit 262 defined by the microfluidic circuitmaterial 260 can comprise multiple channels 264 (two are shown but therecan be more) to which multiple sequestration pens 266 are fluidicallyconnected.

Each sequestration pen 266 can comprise an isolation structure 272, anisolation region 270 within the isolation structure 272, and aconnection region 268. From a proximal opening 274 at the microfluidicchannel 264 to a distal opening 276 at the isolation structure 272, theconnection region 268 fluidically connects the microfluidic channel 264to the isolation region 270. Generally, in accordance with the abovediscussion of FIGS. 2B and 2C, a flow 278 of a first fluidic medium 254in a channel 264 can create secondary flows 282 of the first medium 254from the microfluidic channel 264 into and/or out of the respectiveconnection regions 268 of the sequestration pens 266.

As illustrated in FIG. 2E, the connection region 268 of eachsequestration pen 266 generally includes the area extending between theproximal opening 274 to a channel 264 and the distal opening 276 to anisolation structure 272. The length L_(con) of the connection region 268can be greater than the maximum penetration depth D_(p) of secondaryflow 282, in which case the secondary flow 282 will extend into theconnection region 268 without being redirected toward the isolationregion 270 (as shown in FIG. 2D). Alternatively, at illustrated in FIG.2F, the connection region 268 can have a length L_(con) that is lessthan the maximum penetration depth D_(p), in which case the secondaryflow 282 will extend through the connection region 268 and be redirectedtoward the isolation region 270. In this latter situation, the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than themaximum penetration depth D_(p), so that secondary flow 282 will notextend into isolation region 270. Whether length L_(con) of connectionregion 268 is greater than the penetration depth D_(p), or the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than thepenetration depth D_(p), a flow 278 of a first medium 254 in channel 264that does not exceed a maximum velocity V_(max) will produce a secondaryflow having a penetration depth D_(p), and micro-objects (not shown butcan be the same or generally similar to the micro-objects 246 shown inFIG. 2C) in the isolation region 270 of a sequestration pen 266 will notbe drawn out of the isolation region 270 by a flow 278 of first medium254 in channel 264. Nor will the flow 278 in channel 264 drawmiscellaneous materials (not shown) from channel 264 into the isolationregion 270 of a sequestration pen 266. As such, diffusion is the onlymechanism by which components in a first medium 254 in the microfluidicchannel 264 can move from the microfluidic channel 264 into a secondmedium 258 in an isolation region 270 of a sequestration pen 266.Likewise, diffusion is the only mechanism by which components in asecond medium 258 in an isolation region 270 of a sequestration pen 266can move from the isolation region 270 to a first medium 254 in themicrofluidic channel 264. The first medium 254 can be the same medium asthe second medium 258, or the first medium 254 can be a different mediumthan the second medium 258. Alternatively, the first medium 254 and thesecond medium 258 can start out being the same, then become different,e.g., through conditioning of the second medium by one or more cells inthe isolation region 270, or by changing the medium flowing through themicrofluidic channel 264.

As illustrated in FIG. 2E, the width W_(ch) of the microfluidic channels264 (i.e., taken transverse to the direction of a fluid medium flowthrough the microfluidic channel indicated by arrows 278 in FIG. 2D) inthe microfluidic channel 264 can be substantially perpendicular to awidth W_(con1) of the proximal opening 274 and thus substantiallyparallel to a width W_(con2) of the distal opening 276. The widthW_(con1) of the proximal opening 274 and the width W_(con2) of thedistal opening 276, however, need not be substantially perpendicular toeach other. For example, an angle between an axis (not shown) on whichthe width W_(con1) of the proximal opening 274 is oriented and anotheraxis on which the width W_(con2) of the distal opening 276 is orientedcan be other than perpendicular and thus other than 90°. Examples ofalternatively oriented angles include angles of: about 30° to about 90°,about 45° to about 90°, about 60° to about 90°, or the like.

In various embodiments of sequestration pens (e.g. 124, 126, 128, 130,224, 226, 228, or 266), the isolation region (e.g. 240 or 270) isconfigured to contain a plurality of micro-objects. In otherembodiments, the isolation region can be configured to contain only one,two, three, four, five, or a similar relatively small number ofmicro-objects. Accordingly, the volume of an isolation region can be,for example, at least 1×10⁶, 2×10⁶, 4×10⁶, 6×10⁶ cubic microns, or more.

In various embodiments of sequestration pens, the width W_(ch) of themicrofluidic channel (e.g., 122) at a proximal opening (e.g. 234) can beabout 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns,50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 70-500microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns,70-150 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200microns, 100-150 microns, or 100-120 microns. In some other embodiments,the width W_(ch) of the microfluidic channel (e.g., 122) at a proximalopening (e.g. 234) can be about 200-800 microns, 200-700 microns, or200-600 microns. The foregoing are examples only, and the width W_(ch)of the microfluidic channel 122 can be any width within any of theendpoints listed above. Moreover, the W_(ch) of the microfluidic channel122 can be selected to be in any of these widths in regions of themicrofluidic channel other than at a proximal opening of a sequestrationpen.

In some embodiments, a sequestration pen has a height of about 30 toabout 200 microns, or about 50 to about 150 microns. In someembodiments, the sequestration pen has a cross-sectional area of about1×10⁴-3×10⁶ square microns, 2×10⁴-2×10⁶ square microns, 4×10⁴-1×10⁶square microns, 2×10⁴-5×10⁵ square microns, 2×10⁴-1×10⁵ square micronsor about 2×10⁵-2×10⁶ square microns.

In various embodiments of sequestration pens, the height H_(ch) of themicrofluidic channel (e.g., 122) at a proximal opening (e.g., 234) canbe a height within any of the following heights: 20-100 microns, 20-90microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns,30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns,40-70 microns, 40-60 microns, or 40-50 microns. The foregoing areexamples only, and the height H_(ch) of the microfluidic channel (e.g.,122) can be a height within any of the endpoints listed above. Theheight H_(ch) of the microfluidic channel 122 can be selected to be inany of these heights in regions of the microfluidic channel other thanat a proximal opening of a sequestration pen.

In various embodiments of sequestration pens a cross-sectional area ofthe microfluidic channel (e.g., 122) at a proximal opening (e.g., 234)can be about 500-50,000 square microns, 500-40,000 square microns,500-30,000 square microns, 500-25,000 square microns, 500-20,000 squaremicrons, 500-15,000 square microns, 500-10,000 square microns, 500-7,500square microns, 500-5,000 square microns, 1,000-25,000 square microns,1,000-20,000 square microns, 1,000-15,000 square microns, 1,000-10,000square microns, 1,000-7,500 square microns, 1,000-5,000 square microns,2,000-20,000 square microns, 2,000-15,000 square microns, 2,000-10,000square microns, 2,000-7,500 square microns, 2,000-6,000 square microns,3,000-20,000 square microns, 3,000-15,000 square microns, 3,000-10,000square microns, 3,000-7,500 square microns, or 3,000 to 6,000 squaremicrons. The foregoing are examples only, and the cross-sectional areaof the microfluidic channel (e.g., 122) at a proximal opening (e.g.,234) can be any area within any of the endpoints listed above.

In various embodiments of sequestration pens, the length L_(con) of theconnection region (e.g., 236) can be about 1-600 microns, 5-550 microns,10-500 microns, 15-400 microns, 20-300 microns, 20-500 microns, 40-400microns, 60-300 microns, 80-200 microns, or about 100-150 microns. Theforegoing are examples only, and length L_(con) of a connection region(e.g., 236) can be in any length within any of the endpoints listedabove.

In various embodiments of sequestration pens the width W_(con) of aconnection region (e.g., 236) at a proximal opening (e.g., 234) can beabout 20-500 microns, 20-400 microns, 20-300 microns, 20-200 microns,20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns, 30-400microns, 30-300 microns, 30-200 microns, 30-150 microns, 30-100 microns,30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns, 40-150microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-250 microns,50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-200microns, 60-150 microns, 60-100 microns, 60-80 microns, 70-150 microns,70-100 microns, or 80-100 microns. The foregoing are examples only, andthe width W_(con) of a connection region (e.g., 236) at a proximalopening (e.g., 234) can be different than the foregoing examples (e.g.,any value within any of the endpoints listed above).

In various embodiments of sequestration pens, the width W_(con) of aconnection region (e.g., 236) at a proximal opening (e.g., 234) can beat least as large as the largest dimension of a micro-object (e.g.,biological cell which may be a T cell, or B cell) that the sequestrationpen is intended for. The foregoing are examples only, and the widthW_(con) of a connection region (e.g., 236) at a proximal opening (e.g.,234) can be different than the foregoing examples (e.g., a width withinany of the endpoints listed above).

In various embodiments of sequestration pens, the width W_(pr) of aproximal opening of a connection region may be at least as large as thelargest dimension of a micro-object (e.g., a biological micro-objectsuch as a cell) that the sequestration pen is intended for. For example,the width W_(pr) may be about 50 microns, about 60 microns, about 100microns, about 200 microns, about 300 microns or may be about 50-300microns, about 50-200 microns, about 50-100 microns, about 75-150microns, about 75-100 microns, or about 200-300 microns.

In various embodiments of sequestration pens, a ratio of the lengthL_(con) of a connection region (e.g., 236) to a width W_(con) of theconnection region (e.g., 236) at the proximal opening 234 can be greaterthan or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. Theforegoing are examples only, and the ratio of the length L_(con) of aconnection region 236 to a width W_(con) of the connection region 236 atthe proximal opening 234 can be different than the foregoing examples.

In various embodiments of microfluidic devices 100, 200, 23, 250, 280,290, V_(max) can be set around 0.2, 0.5, 0.7, 1.0, 1.3, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.7, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 11,12, 13, 14, or 15 microliters/sec.

In various embodiments of microfluidic devices having sequestrationpens, the volume of an isolation region (e.g., 240) of a sequestrationpen can be, for example, at least 5×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶,6×10⁶, 8×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, or 8×10⁸ cubic microns, ormore. In various embodiments of microfluidic devices havingsequestration pens, the volume of a sequestration pen may be about5×10⁵, 6×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶, 8×10⁶, 1×10⁷, 3×10⁷, 5×10⁷, orabout 8×10⁷ cubic microns, or more. In some other embodiments, thevolume of a sequestration pen may be about 1 nanoliter to about 50nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2nanoliters to about 10 nanoliters.

In various embodiment, the microfluidic device has sequestration pensconfigured as in any of the embodiments discussed herein where themicrofluidic device has about 5 to about 10 sequestration pens, about 10to about 50 sequestration pens, about 100 to about 500 sequestrationpens; about 200 to about 1000 sequestration pens, about 500 to about1500 sequestration pens, about 1000 to about 2000 sequestration pens,about 1000 to about 3500 sequestration pens, about 3000 to about 7000sequestration pens, about 5000 to about 10,000 sequestration pens, about9,000 to about 15,000 sequestration pens, or about 12,000 to about20,000 sequestration pens. The sequestration pens need not all be thesame size and may include a variety of configurations (e.g., differentwidths, different features within the sequestration pen).

FIG. 2G illustrates a microfluidic device 280 according to oneembodiment. The microfluidic device 280 illustrated in FIG. 2G is astylized diagram of a microfluidic device 100. In practice themicrofluidic device 280 and its constituent circuit elements (e.g.channels 122 and sequestration pens 128) would have the dimensionsdiscussed herein. The microfluidic circuit 120 illustrated in FIG. 2Ghas two ports 107, four distinct channels 122 and four distinct flowpaths 106. The microfluidic device 280 further comprises a plurality ofsequestration pens opening off of each channel 122. In the microfluidicdevice illustrated in FIG. 2G, the sequestration pens have a geometrysimilar to the pens illustrated in FIG. 2C and thus, have bothconnection regions and isolation regions. Accordingly, the microfluidiccircuit 120 includes both swept regions (e.g. channels 122 and portionsof the connection regions 236 within the maximum penetration depth D_(p)of the secondary flow 244) and non-swept regions (e.g. isolation regions240 and portions of the connection regions 236 not within the maximumpenetration depth D_(p) of the secondary flow 244).

FIGS. 3A through 3B shows various embodiments of system 150 which can beused to operate and observe microfluidic devices (e.g. 100, 200, 230,250, 280, 290) according to the present disclosure. As illustrated inFIG. 3A, the system 150 can include a structure (“nest”) 300 configuredto hold a microfluidic device 100 (not shown), or any other microfluidicdevice described herein. The nest 300 can include a socket 302 capableof interfacing with the microfluidic device 320 (e.g., anoptically-actuated electrokinetic device 100) and providing electricalconnections from power source 192 to microfluidic device 320. The nest300 can further include an integrated electrical signal generationsubsystem 304. The electrical signal generation subsystem 304 can beconfigured to supply a biasing voltage to socket 302 such that thebiasing voltage is applied across a pair of electrodes in themicrofluidic device 320 when it is being held by socket 302. Thus, theelectrical signal generation subsystem 304 can be part of power source192. The ability to apply a biasing voltage to microfluidic device 320does not mean that a biasing voltage will be applied at all times whenthe microfluidic device 320 is held by the socket 302. Rather, in mostcases, the biasing voltage will be applied intermittently, e.g., only asneeded to facilitate the generation of electrokinetic forces, such asdielectrophoresis or electro-wetting, in the microfluidic device 320.

As illustrated in FIG. 3A, the nest 300 can include a printed circuitboard assembly (PCBA) 322. The electrical signal generation subsystem304 can be mounted on and electrically integrated into the PCBA 322. Theexemplary support includes socket 302 mounted on PCBA 322, as well.

Typically, the electrical signal generation subsystem 304 will include awaveform generator (not shown). The electrical signal generationsubsystem 304 can further include an oscilloscope (not shown) and/or awaveform amplification circuit (not shown) configured to amplify awaveform received from the waveform generator. The oscilloscope, ifpresent, can be configured to measure the waveform supplied to themicrofluidic device 320 held by the socket 302. In certain embodiments,the oscilloscope measures the waveform at a location proximal to themicrofluidic device 320 (and distal to the waveform generator), thusensuring greater accuracy in measuring the waveform actually applied tothe device. Data obtained from the oscilloscope measurement can be, forexample, provided as feedback to the waveform generator, and thewaveform generator can be configured to adjust its output based on suchfeedback. An example of a suitable combined waveform generator andoscilloscope is the Red Pitaya™.

In certain embodiments, the nest 300 further comprises a controller 308,such as a microprocessor used to sense and/or control the electricalsignal generation subsystem 304. Examples of suitable microprocessorsinclude the Arduino™ microprocessors, such as the Arduino Nano™. Thecontroller 308 may be used to perform functions and analysis or maycommunicate with an external master controller 154 (shown in FIG. 1A) toperform functions and analysis. In the embodiment illustrated in FIG. 3Athe controller 308 communicates with a master controller 154 through aninterface 310 (e.g., a plug or connector).

In some embodiments, the nest 300 can comprise an electrical signalgeneration subsystem 304 comprising a Red Pitaya™ waveformgenerator/oscilloscope unit (“Red Pitaya unit”) and a waveformamplification circuit that amplifies the waveform generated by the RedPitaya unit and passes the amplified voltage to the microfluidic device100. In some embodiments, the Red Pitaya unit is configured to measurethe amplified voltage at the microfluidic device 320 and then adjust itsown output voltage as needed such that the measured voltage at themicrofluidic device 320 is the desired value. In some embodiments, thewaveform amplification circuit can have a +6.5V to −6.5V power supplygenerated by a pair of DC-DC converters mounted on the PCBA 322,resulting in a signal of up to 13 Vpp at the microfluidic device 100.

As illustrated in FIG. 3A, the support structure 300 (e.g., nest) canfurther include a thermal control subsystem 306. The thermal controlsubsystem 306 can be configured to regulate the temperature ofmicrofluidic device 320 held by the support structure 300. For example,the thermal control subsystem 306 can include a Peltier thermoelectricdevice (not shown) and a cooling unit (not shown). The Peltierthermoelectric device can have a first surface configured to interfacewith at least one surface of the microfluidic device 320. The coolingunit can be, for example, a cooling block (not shown), such as aliquid-cooled aluminum block. A second surface of the Peltierthermoelectric device (e.g., a surface opposite the first surface) canbe configured to interface with a surface of such a cooling block. Thecooling block can be connected to a fluidic path 314 configured tocirculate cooled fluid through the cooling block. In the embodimentillustrated in FIG. 3A, the support structure 300 comprises an inlet 316and an outlet 318 to receive cooled fluid from an external reservoir(not shown), introduce the cooled fluid into the fluidic path 314 andthrough the cooling block, and then return the cooled fluid to theexternal reservoir. In some embodiments, the Peltier thermoelectricdevice, the cooling unit, and/or the fluidic path 314 can be mounted ona casing 312 of the support structure 300. In some embodiments, thethermal control subsystem 306 is configured to regulate the temperatureof the Peltier thermoelectric device so as to achieve a targettemperature for the microfluidic device 320. Temperature regulation ofthe Peltier thermoelectric device can be achieved, for example, by athermoelectric power supply, such as a Pololu™ thermoelectric powersupply (Pololu Robotics and Electronics Corp.). The thermal controlsubsystem 306 can include a feedback circuit, such as a temperaturevalue provided by an analog circuit. Alternatively, the feedback circuitcan be provided by a digital circuit.

In some embodiments, the nest 300 can include a thermal controlsubsystem 306 with a feedback circuit that is an analog voltage dividercircuit (not shown) which includes a resistor (e.g., with resistance 1kOhm+/−0.1%, temperature coefficient +/−0.02 ppm/CO) and a NTCthermistor (e.g., with nominal resistance 1 kOhm+/−0.01%). In someinstances, the thermal control subsystem 306 measures the voltage fromthe feedback circuit and then uses the calculated temperature value asinput to an on-board PID control loop algorithm. Output from the PIDcontrol loop algorithm can drive, for example, both a directional and apulse-width-modulated signal pin on a Pololu™ motor drive (not shown) toactuate the thermoelectric power supply, thereby controlling the Peltierthermoelectric device.

The nest 300 can include a serial port 324 which allows themicroprocessor of the controller 308 to communicate with an externalmaster controller 154 via the interface 310 (not shown). In addition,the microprocessor of the controller 308 can communicate (e.g., via aPlink tool (not shown)) with the electrical signal generation subsystem304 and thermal control subsystem 306. Thus, via the combination of thecontroller 308, the interface 310, and the serial port 324, theelectrical signal generation subsystem 304 and the thermal controlsubsystem 306 can communicate with the external master controller 154.In this manner, the master controller 154 can, among other things,assist the electrical signal generation subsystem 304 by performingscaling calculations for output voltage adjustments. A Graphical UserInterface (GUI) (not shown) provided via a display device 170 coupled tothe external master controller 154, can be configured to plottemperature and waveform data obtained from the thermal controlsubsystem 306 and the electrical signal generation subsystem 304,respectively. Alternatively, or in addition, the GUI can allow forupdates to the controller 308, the thermal control subsystem 306, andthe electrical signal generation subsystem 304.

As discussed above, system 150 can include an imaging device 194. Insome embodiments, the imaging device 194 comprises a light modulatingsubsystem 330 (See FIG. 3B). The light modulating subsystem 330 caninclude a digital mirror device (DMD) or a microshutter array system(MSA), either of which can be configured to receive light from a lightsource 332 and transmits a subset of the received light into an opticaltrain of microscope 350. Alternatively, the light modulating subsystem330 can include a device that produces its own light (and thus dispenseswith the need for a light source 332), such as an organic light emittingdiode display (OLED), a liquid crystal on silicon (LCOS) device, aferroelectric liquid crystal on silicon device (FLCOS), or atransmissive liquid crystal display (LCD). The light modulatingsubsystem 330 can be, for example, a projector. Thus, the lightmodulating subsystem 330 can be capable of emitting both structured andunstructured light. In certain embodiments, imaging module 164 and/ormotive module 162 of system 150 can control the light modulatingsubsystem 330.

In certain embodiments, the imaging device 194 further comprises amicroscope 350. In such embodiments, the nest 300 and light modulatingsubsystem 330 can be individually configured to be mounted on themicroscope 350. The microscope 350 can be, for example, a standardresearch-grade light microscope or fluorescence microscope. Thus, thenest 300 can be configured to be mounted on the stage 344 of themicroscope 350 and/or the light modulating subsystem 330 can beconfigured to mount on a port of microscope 350. In other embodiments,the nest 300 and the light modulating subsystem 330 described herein canbe integral components of microscope 350.

In certain embodiments, the microscope 350 can further include one ormore detectors 348. In some embodiments, the detector 348 is controlledby the imaging module 164. The detector 348 can include an eye piece, acharge-coupled device (CCD), a camera (e.g., a digital camera), or anycombination thereof. If at least two detectors 348 are present, onedetector can be, for example, a fast-frame-rate camera while the otherdetector can be a high sensitivity camera. Furthermore, the microscope350 can include an optical train configured to receive reflected and/oremitted light from the microfluidic device 320 and focus at least aportion of the reflected and/or emitted light on the one or moredetectors 348. The optical train of the microscope can also includedifferent tube lenses (not shown) for the different detectors, such thatthe final magnification on each detector can be different.

In certain embodiments, imaging device 194 is configured to use at leasttwo light sources. For example, a first light source 332 can be used toproduce structured light (e.g., via the light modulating subsystem 330)and a second light source 334 can be used to provide unstructured light.The first light source 332 can produce structured light foroptically-actuated electrokinesis and/or fluorescent excitation, and thesecond light source 334 can be used to provide bright fieldillumination. In these embodiments, the motive module 164 can be used tocontrol the first light source 332 and the imaging module 164 can beused to control the second light source 334. The optical train of themicroscope 350 can be configured to (1) receive structured light fromthe light modulating subsystem 330 and focus the structured light on atleast a first region in a microfluidic device, such as anoptically-actuated electrokinetic device, when the device is being heldby the nest 300, and (2) receive reflected and/or emitted light from themicrofluidic device and focus at least a portion of such reflectedand/or emitted light onto detector 348. The optical train can be furtherconfigured to receive unstructured light from a second light source andfocus the unstructured light on at least a second region of themicrofluidic device, when the device is held by the nest 300. In certainembodiments, the first and second regions of the microfluidic device canbe overlapping regions. For example, the first region can be a subset ofthe second region. In other embodiments, the second light source 334 mayadditionally or alternatively include a laser, which may have anysuitable wavelength of light. The representation of the optical systemshown in FIG. 3B is a schematic representation only, and the opticalsystem may include additional filters, notch filters, lenses and thelike. When the second light source 334 includes one or more lightsource(s) for brightfield and/or fluorescent excitation, as well aslaser illumination the physical arrangement of the light source(s) mayvary from that shown in FIG. 3B, and the laser illumination may beintroduced at any suitable physical location within the optical system.The schematic locations of light source 334 and light source 332/lightmodulating subsystem 330 may be interchanged as well.

In FIG. 3B, the first light source 332 is shown supplying light to alight modulating subsystem 330, which provides structured light to theoptical train of the microscope 350 of system 355 (not shown). Thesecond light source 334 is shown providing unstructured light to theoptical train via a beam splitter 336. Structured light from the lightmodulating subsystem 330 and unstructured light from the second lightsource 334 travel from the beam splitter 336 through the optical traintogether to reach a second beam splitter (or dichroic filter 338,depending on the light provided by the light modulating subsystem 330),where the light gets reflected down through the objective 336 to thesample plane 342. Reflected and/or emitted light from the sample plane342 then travels back up through the objective 340, through the beamsplitter and/or dichroic filter 338, and to a dichroic filter 346. Onlya fraction of the light reaching dichroic filter 346 passes through andreaches the detector 348.

In some embodiments, the second light source 334 emits blue light. Withan appropriate dichroic filter 346, blue light reflected from the sampleplane 342 is able to pass through dichroic filter 346 and reach thedetector 348. In contrast, structured light coming from the lightmodulating subsystem 330 gets reflected from the sample plane 342, butdoes not pass through the dichroic filter 346. In this example, thedichroic filter 346 is filtering out visible light having a wavelengthlonger than 495 nm. Such filtering out of the light from the lightmodulating subsystem 330 would only be complete (as shown) if the lightemitted from the light modulating subsystem did not include anywavelengths shorter than 495 nm. In practice, if the light coming fromthe light modulating subsystem 330 includes wavelengths shorter than 495nm (e.g., blue wavelengths), then some of the light from the lightmodulating subsystem would pass through filter 346 to reach the detector348. In such an embodiment, the filter 346 acts to change the balancebetween the amount of light that reaches the detector 348 from the firstlight source 332 and the second light source 334. This can be beneficialif the first light source 332 is significantly stronger than the secondlight source 334. In other embodiments, the second light source 334 canemit red light, and the dichroic filter 346 can filter out visible lightother than red light (e.g., visible light having a wavelength shorterthan 650 nm).

Coating Solutions and Coating Agents.

Without intending to be limited by theory, maintenance of a biologicalmicro-object (e.g., a biological cell) within a microfluidic device(e.g., a DEP-configured and/or EW-configured microfluidic device) may befacilitated (i.e., the biological micro-object exhibits increasedviability, greater expansion and/or greater portability within themicrofluidic device) when at least one or more inner surfaces of themicrofluidic device have been conditioned or coated so as to present alayer of organic and/or hydrophilic molecules that provides the primaryinterface between the microfluidic device and biological micro-object(s)maintained therein. In some embodiments, one or more of the innersurfaces of the microfluidic device (e.g. the inner surface of theelectrode activation substrate of a DEP-configured microfluidic device,the cover of the microfluidic device, and/or the surfaces of the circuitmaterial) may be treated with or modified by a coating solution and/orcoating agent to generate the desired layer of organic and/orhydrophilic molecules.

The coating may be applied before or after introduction of biologicalmicro-object(s), or may be introduced concurrently with the biologicalmicro-object(s). In some embodiments, the biological micro-object(s) maybe imported into the microfluidic device in a fluidic medium thatincludes one or more coating agents. In other embodiments, the innersurface(s) of the microfluidic device (e.g., a DEP-configuredmicrofluidic device) are treated or “primed” with a coating solutioncomprising a coating agent prior to introduction of the biologicalmicro-object(s) into the microfluidic device.

In some embodiments, at least one surface of the microfluidic deviceincludes a coating material that provides a layer of organic and/orhydrophilic molecules suitable for maintenance and/or expansion ofbiological micro-object(s) (e.g. provides a conditioned surface asdescribed below). In some embodiments, substantially all the innersurfaces of the microfluidic device include the coating material. Thecoated inner surface(s) may include the surface of a flow region (e.g.,channel), chamber, or sequestration pen, or a combination thereof. Insome embodiments, each of a plurality of sequestration pens has at leastone inner surface coated with coating materials. In other embodiments,each of a plurality of flow regions or channels has at least one innersurface coated with coating materials. In some embodiments, at least oneinner surface of each of a plurality of sequestration pens and each of aplurality of channels is coated with coating materials.

Coating Agent/Solution.

Any convenient coating agent/coating solution can be used, including butnot limited to: serum or serum factors, bovine serum albumin (BSA),polymers, detergents, enzymes, and any combination thereof.

Polymer-Based Coating Materials.

The at least one inner surface may include a coating material thatcomprises a polymer. The polymer may be covalently or non-covalentlybound (or may be non-specifically adhered) to the at least one surface.The polymer may have a variety of structural motifs, such as found inblock polymers (and copolymers), star polymers (star copolymers), andgraft or comb polymers (graft copolymers), all of which may be suitablefor the methods disclosed herein.

The polymer may include a polymer including alkylene ether moieties. Awide variety of alkylene ether containing polymers may be suitable foruse in the microfluidic devices described herein. One non-limitingexemplary class of alkylene ether containing polymers are amphiphilicnonionic block copolymers which include blocks of polyethylene oxide(PEO) and polypropylene oxide (PPO) subunits in differing ratios andlocations within the polymer chain. Pluronic® polymers (BASF) are blockcopolymers of this type and are known in the art to be suitable for usewhen in contact with living cells. The polymers may range in averagemolecular mass M_(w) from about 2000 Da to about 20 KDa. In someembodiments, the PEO-PPO block copolymer can have ahydrophilic-lipophilic balance (HLB) greater than about 10 (e.g. 12-18).Specific Pluronic® polymers useful for yielding a coated surface includePluronic® L44, L64, P85, and F127 (including F127NF). Another class ofalkylene ether containing polymers is polyethylene glycol (PEGM_(w)<100,000 Da) or alternatively polyethylene oxide (PEO,M_(w)>100,000). In some embodiments, a PEG may have an M_(w) of about1000 Da, 5000 Da, 10,000 Da or 20,000 Da.

In other embodiments, the coating material may include a polymercontaining carboxylic acid moieties. The carboxylic acid subunit may bean alkyl, alkenyl or aromatic moiety containing subunit. Onenon-limiting example is polylactic acid (PLA). In other embodiments, thecoating material may include a polymer containing phosphate moieties,either at a terminus of the polymer backbone or pendant from thebackbone of the polymer. In yet other embodiments, the coating materialmay include a polymer containing sulfonic acid moieties. The sulfonicacid subunit may be an alkyl, alkenyl or aromatic moiety containingsubunit. One non-limiting example is polystyrene sulfonic acid (PSSA) orpolyanethole sulfonic acid. In further embodiments, the coating materialmay include a polymer including amine moieties. The polyamino polymermay include a natural polyamine polymer or a synthetic polyaminepolymer. Examples of natural polyamines include spermine, spermidine,and putrescine.

In other embodiments, the coating material may include a polymercontaining saccharide moieties. In a non-limiting example,polysaccharides such as xanthan gum or dextran may be suitable to form amaterial which may reduce or prevent cell sticking in the microfluidicdevice. For example, a dextran polymer having a size about 3 kDa may beused to provide a coating material for a surface within a microfluidicdevice.

In other embodiments, the coating material may include a polymercontaining nucleotide moieties, i.e. a nucleic acid, which may haveribonucleotide moieties or deoxyribonucleotide moieties, providing apolyelectrolyte surface. The nucleic acid may contain only naturalnucleotide moieties or may contain unnatural nucleotide moieties whichcomprise nucleobase, ribose or phosphate moiety analogs such as7-deazaadenine, pentose, methyl phosphonate or phosphorothioate moietieswithout limitation.

In yet other embodiments, the coating material may include a polymercontaining amino acid moieties. The polymer containing amino acidmoieties may include a natural amino acid containing polymer or anunnatural amino acid containing polymer, either of which may include apeptide, a polypeptide or a protein. In one non-limiting example, theprotein may be bovine serum albumin (BSA) and/or serum (or a combinationof multiple different sera) comprising albumin and/or one or more othersimilar proteins as coating agents. The serum can be from any convenientsource, including but not limited to fetal calf serum, sheep serum, goatserum, horse serum, and the like. In certain embodiments, BSA in acoating solution is present in a concentration from about 1 mg/mL toabout 100 mg/mL, including 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or more oranywhere in between. In certain embodiments, serum in a coating solutionmay be present in a concentration of about 20% (v/v) to about 50% v/v,including 25%, 30%, 35%, 40%, 45%, or more or anywhere in between. Insome embodiments, BSA may be present as a coating agent in a coatingsolution at 5 mg/mL, whereas in other embodiments, BSA may be present asa coating agent in a coating solution at 70 mg/mL. In certainembodiments, serum is present as a coating agent in a coating solutionat 30%. In some embodiments, an extracellular matrix (ECM) protein maybe provided within the coating material for optimized cell adhesion tofoster cell growth. A cell matrix protein, which may be included in acoating material, can include, but is not limited to, a collagen, anelastin, an RGD-containing peptide (e.g. a fibronectin), or a laminin.In yet other embodiments, growth factors, cytokines, hormones or othercell signaling species may be provided within the coating material ofthe microfluidic device.

In some embodiments, the coating material may include a polymercontaining more than one of alkylene oxide moieties, carboxylic acidmoieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, or amino acid moieties. In otherembodiments, the polymer conditioned surface may include a mixture ofmore than one polymer each having alkylene oxide moieties, carboxylicacid moieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, and/or amino acid moieties, which may beindependently or simultaneously incorporated into the coating material.

In addition, in embodiments in which a covalently modified surface isused in conjunction with coating agents, the anions, cations, and/orzwitterions of the covalently modified surface can form ionic bonds withthe charged portions of non-covalent coating agents (e.g. proteins insolution) that are present in a fluidic medium (e.g. a coating solution)in the enclosure.

Further details of appropriate coating treatments and modifications maybe found at U.S. application Ser. No. 15/135,707, filed on Apr. 22,2016, and is incorporated by reference in its entirety.

Additional System Components for Maintenance of Viability of Cellswithin the Sequestration Pens of the Microfluidic Device.

In order to promote growth and/or expansion of cell populations,environmental conditions conducive to maintaining functional cells maybe provided by additional components of the system. For example, suchadditional components can provide nutrients, cell growth signalingspecies, pH modulation, gas exchange, temperature control, and removalof waste products from cells.

Kits.

In various embodiments, a kit for providing a microfluidic device havingat least one covalently modified surface configured to supportbiological cell growth, viability and/or portability includes: amicrofluidic device comprising an enclosure comprising a base, a cover,and microfluidic circuit material defining a fluidic circuit therein,wherein at least one inner surface of the base, the cover and themicrofluidic circuit material has a first covalently bound surfacemodification comprising a first linking group, and a first moiety,wherein the first moiety is a first surface contact moiety or a firstreactive moiety; wherein at least one inner surface of the base, thecover and the microfluidic circuit material has a second covalentlybound surface modification comprising a second linking group, and asecond moiety, wherein the second moiety is a second surface contactmoiety or second reactive moiety, and wherein the first linking groupand the second linking group are different from each other and/or thefirst moiety is different from the second moiety.

The first covalently bound surface modification and the secondcovalently bound surface modification of the microfluidic device of thekit, may each have a structure independently selected from Formula XXX,Formula V, Formula VII, Formula XXXI, Formula VIII, and Formula IX

wherein LG is —W—Si(OZ)₂O— or —OP(O)₂O—; L_(fm) is a linker comprising 1to 200 non-hydrogen atoms selected from any combination of silicon,carbon, nitrogen, oxygen, sulfur and phosphorus atoms and furthercomprises 0 or 1 coupling groups CG; R_(x) is a reactive moiety; W is O,S, or N, Z is a bond to an adjacent silicon atom or is a bond to thesurface; n is an integer of 3-21, L_(sm) is a linker comprising 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms and further comprises 0,1, 2, 3, or 4 coupling groups CG; and

is the surface. Kits may be provided including any microfluidic deviceas described herein.

The kit may further include a surface modifying reagent, which has astructure of Formula XII:

RP-L-surface contact moiety  Formula XII

where RP is a reaction pair moiety; L is a linker and surface contactmoiety is a moiety that provides improved contact characteristics forbiological micro-objects. L is a linker; wherein L comprises a bond or 1to 200 non-hydrogen atoms selected from any combination of silicon,carbon, nitrogen, oxygen, sulfur and phosphorus atoms, and comprises 0,1, 2, or 3 coupling groups CG. Surface contact moiety is as definedabove f. L and surface contact moiety may have any combination in thesurface modifying reagent. In some embodiments, the surface contactmoiety may include polyethylene glycol. In other embodiments, thesurface contact moiety may include dextran. The reaction pair moiety isconfigured to react, respectively, with the reactive moiety of afunctionalized surface.

The kit may further include a secondary functionalizing reagent having astructure of Formula XXXIV:

RP-L_(fm)-R_(x2)  Formula XXXIV,

wherein RP is a reaction pair moiety for reacting with the reactivemoiety of Formula XXX, Formula V, or Formula VII; and L_(fm) is a linkercomprising 1 to 200 non-hydrogen atoms selected from any combination ofsilicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms andfurther comprises 0 or 1 coupling groups CG. R_(x2) is selected to notreact with the reactive moiety of the functionalized surface.

The kit may further include other reagents to be used in producing amicrofluidic device having at least one covalently modified surface ofFormula VIII. Suitable reaction media, buffers, or reaction accelerantsmay be provided in the kit. The auxiliary reagents and/or surfacemodifying reagent and/or secondary functionalizing reagent may beprovided in separate containers.

Synthesis of the Compound of Formula IV.

A method of synthesizing a compound having a structure of Formula VI isprovided, including the step of reacting a compound of Formula XIII withazide ion, and producing the compound of Formula VI as shown in Equation2, where h is 1 to 19, n is 3 to 21, and R is H or C₁-C₆ alkyl. In someembodiments, n is an integer of 7 to 21.

The azide ion may be provided as sodium azide or any other suitablesource of azide ion. The reaction may be performed in any suitablesolvent such as acetonitrile or DMF. The reaction may be performed atambient temperature, which may be in a range of about 15° C. to about30° C. In some embodiments, an ambient reaction temperature may be in arange of about 20° C. to about 30° C. In some embodiments, the reactionmay be performed at a temperature selected from 30° C., 40° C., 50° C.,60° C., or 70° C. The reaction may be performed under an inertatmosphere.

In other embodiments, a compound having a structure of Formula XIII:

is provided where h is an integer of 1 to 19 and R is selectedindependently from the group consisting of H and C₁-C₆ alkyl. Thecompound of Formula II may be a useful starting material for thesynthesis of the compound having a structure of Formula I. In someembodiments, h may be 5 to 19. In other embodiments, h may be 7 to 21, 8to 21, 9 to 21, 10 to 21, 11 to 21, 12 to 21, 13 to 21, 14 to 21, 15 to21, or 16 to 21. In other embodiments, h may be 7, 12, or 14. In someembodiments, h may be 10, 12, or 14. In some embodiments h may be 12 or14. In various embodiments, each instance of R may be Me or Et.

Synthesis of the Compound of Formula XIII.

A method of synthesizing a compound having a structure of Formula XIIIis provided including the step of: reacting an olefinic compound(compound 1) with a silane (compound 2), in the presence of a catalystor an initiator, thereby producing the compound having a structure ofFormula XIII. (See Equation 3)

For the compounds 1, 2, and Formula XIII of Equation 3, h is an integerof 1 to 19 and each instance of R is independently H or C₁-C₆ alkyl. Insome embodiments, h is an integer of about 5 to 19. In some embodiments,R is C₁-C₆ alkyl.

In some embodiments, each instance of R may be selected from methyl,ethyl and propyl. In other embodiments, each instance of R may bemethyl. In various embodiments, h may be 7 to 19. In other embodiments,h may be 7, 12, or 14. In some embodiments, h may be 7.

In some embodiments, the catalyst may be any suitable hydrosilylationcatalyst. The catalyst may be a transition metal complex M(L)_(n) whereL is a ligand, and M is a metal such as Fe(0), Co(I), Rh(I), Ni(0),Pd(0), Pt(II) or Pt(0). In some embodiments, the metal of thehydrosilylation catalyst complex may be Co(I), Rh(I), Ni(0), Pt(II) orPt(0). In yet other embodiments, the metal may be Rh(I), Pt(II), orPt(0). The ligands may be selected to create an electron rich complexand may be any suitable ligand. Ligands may include halogen (e.g.,chlorine); olefins, nitriles, siloxanes (including simpletetraalkyldivinyl siloxanes or constrained SILOP ligands; aromaticmoieties 2,2′-bis(diphenylphosphino)-sterically constrained biphenyl orbinaphthyl ligands such as BINAP, BIPHEP, BINEPINE, or PHANEPHOS;2,3-O-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butyl(DIOP); (Some examples of SUITABLE HYDROSILYLATION CATALYSTS CAN INCLUDEBUT ARE NOT LIMITED TO H₂PTCL₆.6H₂O/IPROH (SPEIER catalyst); chloro(1,5-cyclooctadiene) Rh(1) dimer ([Rh(cod)Cl]2;Tris(triphenylphosphine)-rhodium(I) chloride; [PtCl₂(NCR)₂ where R maybe N(alkyl)₂, particularly methyl or a cyclic amine such asN-piperidinyl, Ph, Ch₂Ph; Karsted's catalyst Pt₂{[(CH₂═CH₂)SiMe₂]O}₃);and bis(imino) pyridine iron dinitrogen complexes (^(et) PDI)Fe(N₂)]₂(mu₂-azido).

In some embodiments, the catalyst may be a platinum (0) catalyst. Theplatinum (0) catalyst may be aPt(0)-1,1,3,3-tetramethyl-1,3-divinyldisiloxane complex (Cpd. 3):

In other embodiments, an initiator may be used, which may be present ina range of about 0.5 equivalents to about 1.4 equivalents. In someembodiments, the initiator may be trialkylborane.

The reaction may be performed in any solvent that is capable ofdissolving the olefinic compound (Cpd. 1 of Equation 2), which mayinclude but is not limited to DMF, benzene, tetrahydrofuran, toluene, 1,3-bis trifluoromethyl benzene, amongst other fluorinated or partiallyfluorinated solvents. In some embodiments, toluene or dimethyl formamide(DMF) may be used.

The reaction may be performed under an inert atmosphere, which may beargon or nitrogen gas. Typically, an inert atmosphere will exclude watervapor.

Elevated temperatures may be used to promote reaction, and the reactionmay be performed at a temperature in a range of about 60° C. to about110° C. In some embodiments, the reaction may be performed at about 60°C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C.,105° C., or about 110° C. The reaction may be completed after about 6 h,10 h, 14 h, 18 h, 24 h, 30 h, 48 h, 60 h or any time point in between.

In some other embodiments, a method of synthesizing a compound having astructure of Formula IV:

may be provided, which includes the step of reacting a compound having astructure of Formula XIV:

with an alcohol ROH, in the presence of a base, wherein h is an integerof 1 to 19; each instance of X is Cl; ROH is methyl alcohol, ethylalcohol, or propyl alcohol, thereby producing the compound of Formula I,wherein h is an integer of 1 to 19, and R is C₁-C₃ alkyl. In someembodiments, the base may be pyridine. In some embodiments, h is aninteger of 5 to 19. In various embodiments, h may be 7, 12 or 14. In yetother embodiments, R may be methyl.

Synthesis of the Compound of Formula L.

A method of synthesizing a compound having a structure of Formula L isprovided, including the step of: reacting an olefinic compound(compound 1) with a silane (compound 2), in the presence of a catalystor an initiator, thereby producing the compound having a structure ofFormula L (See Equation 1).

For the compounds 101, 1032, and Formula L of Equation 4, n may be aninteger of 13 to 25; each instance of Y may be independently halo, OH,or OR, halo is Br, Cl or F; and R is C₁-C₆ alkyl. In some embodiments,each instance of Y is Cl. In other embodiments, each instance of Y maybe methoxy, ethoxy, or propoxy. In various embodiments, n may be 13, 15,17, or 19. In some embodiments, n may be 13 or 15.

In some embodiments, the compound of Formula L may be a compound havinga structure of Formula LI, and may be produced according to the methodshown in Equation 5. The olefinic compound (compound 1) may be reactedwith a silane having three substituents OR (compound 3) where R may be Hor C₁ to C₆ alkyl. In some embodiments, each instance of R may beselected from methyl, ethyl and propyl. In other embodiments, eachinstance of R may be methyl. In various embodiments, n may be 13, 15,17, or 19. In some embodiments, n may be 13 or 15.

In other embodiments, the compound of Formula L may be a compound havinga structure of Formula LII, and may be produced according to the methodshown in Equation 6.

The olefinic compound, 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 9, 9, 10, 10,11, 11, 12, 12, 13, 13, 14, 14, 15, 15, 16, 16,16-nonacosafluorohexadec-1-ene (Cpd. 105) may be reacted with atrialkoxysilane (Cpd. 104) in the presence of a catalyst or initiator;thereby producing the molecule of Formula LII. The catalyst may be anysuitable hydrosilylation catalyst.

Synthesis of the Compound of Formula VI.

The compound of Formula VI may be synthesized by various routes, one ofwhich may include reacting the compound of Formula IV with a metalacetylide.

EXAMPLES

System and Microfluidic Device:

An OptoSelect chip, a nanofluidic device manufactured by BerkeleyLights, Inc. and controlled by an optical instrument which was alsomanufactured by Berkeley Lights, Inc. The instrument included: amounting stage for the chip coupled to a temperature controller; a pumpand fluid medium conditioning component; and an optical train includinga camera and a structured light source suitable for activatingphototransistors within the chip. The OptoSelect™ chip included asubstrate configured with OptoElectroPositioning (OEP™) technology,which provides a phototransistor-activated OET force. The chip alsoincluded a plurality of microfluidic channels, each having a pluralityof NanoPen™ chambers (or sequestration pens) fluidically connectedthereto. The volume of each sequestration pen was around 1×10⁶ cubicmicrons.

Priming Solution:

Complete growth medium containing 0.1% Pluronic® F127 ((LifeTechnologies® Cat# P6866).

Preparation for culturing: The microfluidic device having a modifiedsurface was loaded onto the system and purged with 100% carbon dioxideat 15 psi for 5 min. Immediately following the carbon dioxide purge, thepriming solution was perfused through the microfluidic device at 5microliters/sec for 8 min. Culture medium was then flowed through themicrofluidic device at 5 microliters/sec for 5 min.

Priming Regime.

250 microliters of 100% carbon dioxide was flowed in at a rate of 12microliters/sec. This was followed by 250 microliters of PBS containing0.1% Pluronic® F27 (Life Technologies® Cat# P6866), flowed in at 12microliters/sec. The final step of priming included 250 microliters ofPBS, flowed in at 12 microliters/sec. Introduction of the culture mediumfollows.

Perfusion Regime.

The perfusion method was either of the following two methods:

1. Perfuse at 0.01 microliters/sec for 2 h; perfuse at 2 microliters/secfor 64 sec; and repeat.

2. Perfuse at 0.02 microliters/sec for 100 sec; stop flow 500 sec;perfuse at 2 microliters/sec for 64 sec; and repeat.

Example 1. Synthesis of (11-bromoundecyl) trimethoxysilane

(Compound 4) 1.26 g (5.4 mmol) of 11-bromoundec-1-ene (Sigma Aldrich) issolubilized in 150 ml of dry toluene (Oakwood Products) in an argonflushed reaction vessel equipped with a reflux condensorTrimethoxysilane (1.77 g, 14.5 mmol, Sigma Aldrich Cat. No. 282626) isadded to the reaction under argon purging via syringe through a septum.Next, 1.5 g of hydrosilation catalyst solution, (0.08 mmol) ofhydrosilation catalyst platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex (Compound 3, 0.1Min poly(dimethylsiloxane), Sigma Aldrich, Cat. No. 479527) is added tothe reaction under argon purging via syringe. The reaction is thencontinued under an argon atmosphere at a temperature of 80° C. for 24hours to produce (11-bromoundecyl) trimethyoxysilane (Compound 4). Thereaction is allowed to cool to room temperature under argon, filtered,and the product is extracted into pentane and the solvent is removed byrotary evaporation at reduced pressure. The product is purified byvacuum distillation.

Example 2. Synthesis of (11-azidoundecyl)trimethoxysilane

(Compound 5). (13-11-azidoundecyltrimethoxysilane was synthesized from11-bromoundecyltrimethoxysilane (Gelest) by displacing the bromides withsodium azide. In a typical reaction, 4.00 g of11-bromoundecyltrimethoxysilane (Gelest Cat. # SIB 1908.0) was added toa solution containing 2.00 g of sodium azide (Sigma-Aldrich) in 60 mL ofdry dimethylformamide (Acros). The solution was stirred for 24 h at roomtemperature under nitrogen. Next, the solution was filtered, and thefiltrate was extracted with dry pentane (Acros). The crude11-azidoundecyltrimethoxysilane product was concentrated by rotaryevaporation and was purified by two successive vacuum distillations andcharacterized using NMR and FTIR spectroscopies.

Example 3. Preparation of a Functionalized Surface of a Silicon Wafer

A silicon wafer (780 microns thick, 1 cm by 1 cm) was treated in anoxygen plasma cleaner (Nordson Asymtek) for 5 min, using 100 W power,240 mTorr pressure and 440 sccm oxygen flow rate. The plasma treatedsilicon wafer was treated in a vacuum reactor with (11-azidoundecyl)trimethoxy silane (Compound 5, 300 microliters) in a foil boat in thebottom of the vacuum reactor in the presence of magnesium sulfateheptahydrate (0.5 g, Acros Cat. #10034-99-8), as a water reactant sourcein a separate foil boat in the bottom of the vacuum reactor. The chamberwas then pumped to 750 mTorr using a vacuum pump and then sealed. Thevacuum reactor was placed within an oven heated at 110° C. for 24-48 h.This introduced a modified surface to the wafer, where thefunctionalized surface had a structure of Formula XV:

where Z is a bond to an adjacent silicon atom bound to the surface or isa bond to the surface and

is the surface. After cooling to room temperature and introducing argonto the evacuated chamber, the wafer was removed from the reactor. Thewafer was rinsed with acetone, isopropanol, and dried under a stream ofnitrogen. Confirmation of introduction of the functionalized surface wasmade by ellipsometry and contact angle goniometry.

Example 4. Modification of Microfluidic Circuit Material

One example of microfluidic circuit material is photopatternablesilicone and was used to define the fluidic circuit within themicrofluidic device. Proof of modification of this material wasobtained. An ITO wafer having photopatterned silicone structuresincorporated upon it was in an oxygen plasma cleaner (Nordson Asymtek)for 5 min, using 100 W power, 240 mTorr pressure and 440 sccm oxygenflow rate. The plasma-cleaned photopatterned ITO wafer was treated asdescribed in Example 3 to introduce a modified surface of Formula XVonto the photopatterned silicone. FTIR ATR (attenuated totalreflectance) spectra were measured using a ThermoFisher Nicolet iS50spectrometer with a liquid nitrogen cooled MCT detector. Spectra werecollected on a Harrick Vari-GATR accessory by pressing thephotopatterned silicone, modified with a surface of Formula XV, againstthe surface of the germanium crystal with 200 N force. In pressing themodified photopatternable silicone material against the germaniumcrystal, FTIR ATR was obtained only of the modified photopatternedsilicone. 250 scans were collected at 4 cm-1 resolution and referencedagainst a background spectrum of the bare Germanium crystal. Spectrawere visualized using Omnic software provided with the FTIRspectrometer.

As shown in FIG. 4, the peak at ˜2098 cm-1 (410) is attributable to theazide asymmetric stretch. Peaks at 2924 cm-1 (414) and 2854 cm-1 (412)are attributable to C—H stretching modes

Note:

In the following examples of introduction of a modified surface to amicrofluidic device, the contact angle and thickness measurements wereperformed on silicon wafers modified in the same manner as the specificmodified surface in the microfluidic device.

Example 5. Preparation of a Microfluidic Device Having Modified InteriorSurfaces of Formula XV

An OptoSelect device having a first silicon electrode activationsubstrate and a second ITO substrate on the opposite wall, andphotopatterned silicone microfluidic circuit material separating the twosubstrates, was treated in an oxygen plasma cleaner (Nordson Asymtek)for 1 min, using 100 W power, 240 mTorr pressure and 440 sccm oxygenflow rate. The plasma treated microfluidic device was treated in avacuum reactor with 3-azidoundecyl) trimethoxysilane (Compound 5, 300microliters) in a foil boat in the bottom of the vacuum reactor in thepresence of magnesium sulfate heptahydrate (0.5 g, Acros), as a waterreactant source in a separate foil boat in the bottom of the vacuumreactor. The chamber was then pumped to 750 mTorr using a vacuum pumpand then sealed. The vacuum reactor was placed within an oven heated at110° C. for 24-48 h. This introduced a functionalized surface to all ofthe inner facing surfaces of the microfluidic device, where thefunctionalized surface had a structure of Formula XV:

where Z is a bond to an adjacent silicon atom bound to the surface or isa bond to the surface and

is the surface. After cooling to room temperature and introducing argonto the evacuated chamber, the microfluidic device was removed from thereactor. The microfluidic device having the functionalized surface wasthen treated with an alkynyl species to introduce the desired modifiedsurface as described below in Examples 6 and 7.

Example 6. Introduction of a Polyethylene Glycol (PEG) Modified Surface(Formula XVI) to a Microfluidic Device

Materials. Alkyne-modified PEG (j=MW ˜5000 Da)(Compound 6) was purchasedfrom JenKem and used as received.

Sodium ascorbate and copper sulfate pentahydrate were purchased fromSigma-Aldrich and used as received. (3[tris(3-hydroxypropyltriazolylmethyl)amine) THPTA rate acceleratingligand (Glen Research) was used as received.

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was reacted with alkyne-modified PEG(Compound 5) by flowing at least 250 microliters of an aqueous solutioncontaining 3.3 millimolar alkyne-modified PEG, 50 millimolar coppersulfate, 55 millimolar THPTA ligand and 100 millimolar sodium ascorbatethrough the microfluidic devices having the 11-azidoundecylsiloxysurface modifying ligand. The reaction was allowed to proceed at roomtemperature for at least 1 hour. The microfluidic device having a PEGmodified surface of Formula XVI:

where Z is as defined above for Formula VIII, and

is the surface, was then rinsed by flowing at least 250 microliters ofdeionized water through the devices. After completion of the cyclizationreaction that introduces the modified surface, the thickness of thelayer increased from 1.4 nm (functionalized surface thickness) to 5 nmin thickness. Additionally, the sessile drop water contact angledecreased from approximately 80 degrees (functionalized surface ofFormula XV) to 35 degrees (surface of Formula XVI).

Example 7. Introduction of a Dextran Modified Surface (Formula XVII) toa Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV as described above, was treated with dibenzylcyclooctynyl(DBCO) modified-dextran, weight averaged molecular weight 3000 Da(Compound 7, Nanocs):

by flowing at least 250 microliters of an aqueous solution containing1.66 millimolar DBCO-dextran through the microfluidic devices havingsurface modifying azide ligands after vapor deposition. The reaction wasallowed to proceed at room temperature for at least 1 h. Themicrofluidic device having a modified surface of Formula XVII (one oftwo regioisomers shown):

where Z is as defined above for Formula VIII, and

is the surface, was then rinsed by flowing at least 250 microliters ofDI water through the chips.

Example 8. Alternative Introduction of a Polyethylene Glycol (PEG)Modified Surface (Formula XVIII) to a Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV as described above, was treated with dibenzylcyclooctynyl(DBCO) modified-PEG, weight averaged molecular weight 5000 Da (Compound8, Broadpharm, Cat. # BP-22461) by flowing at least 250 microliters ofan aqueous solution containing 1.33 millimolar DBCO-PEG through themicrofluidic device having surface modifying azide ligands after vapordeposition. The reaction was allowed to proceed at 40° C. for at least 1h. The microfluidic device having a modified surface of Formula XVIIIwas then rinsed by flowing at least 250 microliters of DI water throughthe chips. One of two regioisomers shown.

Example 9. Introduction of a Poly-L-Glutamic Acid (PGA) Modified Surface(Formula XIX) to a Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was reacted with alkyne-modified poly(L-glutamic acid/salt)(PGA, MW=15,000 Da)(Compound 8, Alamanda™Polymers, Cat. # AK-PLE100):

(where (*) is a proprietary linker) by flowing at least 250 microlitersof a buffered saline solution (5.4×PBS pH 7.4) containing 1.33millimolar alkyne-modified PGA, 500 micromolar copper sulfate, 550micromolar THPTA ligand and 5 millimolar sodium ascorbate through themicrofluidic devices having the 11-azidoundecylsiloxy surface modifyingligand. The reaction was allowed to proceed at room temperature for atleast 1 hour or 40° C. for at least 15 minutes. The microfluidic devicehaving a PGA modified surface of Formula XIV was then rinsed by flowingat least 250 microliters of deionized water through the devices. Aftercompletion of the cyclization reaction that introduces the modifiedsurface, the thickness of the layer increased from 1.1 nm(functionalized surface thickness) to 5.2 nm in thickness. Additionally,the sessile drop water contact angle decreased from approximately 80degrees (functionalized surface of Formula X) to 17 degrees (surface ofFormula XIX).

Example 10. Introduction of a Covalently Modified Surface of BiotinFunctionalized PEG Surface (Formula XX) to a Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was reacted with biotin functionalizedalkynyl PEG (PEG is 1 kDA, Compound 9, Nanocs, Cat. #PG2-AKBN-1k):

by flowing at least 250 microliters of an aqueous solution containing31.33 millimolar Compound 9, 500 micromolar copper sulfate, 550micromolar THPTA ligand and 5 millimolar sodium ascorbate through themicrofluidic devices having the 11-azidoundecylsiloxy surface modifyingligand (Formula XV). The reaction was allowed to proceed at roomtemperature for at least 1 hour. The microfluidic device having abiotinylated PEG modified surface of Formula XX

was then rinsed by flowing at least 250 microliters of deionized waterthrough the devices. After completion of the cyclization reaction thatintroduces the modified surface, the thickness of the layer increasedfrom 1.4 nm (functionalized surface thickness) to 5 nm in thickness.Additionally, the sessile drop water contact angle decreased fromapproximately 80 degrees (functionalized surface of Formula XV) to 39degrees (surface of Formula XX).

Example 11. Introduction of a Covalently Modified Surface ofPhotocleavable Biotin Functionalized PEG Surface (Formula XXI) to aMicrofluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was reacted with biotin functionalizedphotocleavable alkyne PEG3 (Compound 10, Broadpharm, Cat. # BP-22677,which contains the photocleavable nitro substituted phenyl group as partof the linker L), flowing at least 250 microliters of an aqueoussolution containing 1.33 millimolar Compound 10, 500 micromolar coppersulfate, 550 micromolar THPTA ligand and 5 millimolar sodium ascorbatethrough the microfluidic devices having the 11-azidoundecylsiloxysurface modifying ligand. The reaction was allowed to proceed at roomtemperature for at least 1 hour. The microfluidic device having abiotinylated PEG modified surface of Formula XX1:

was then rinsed by flowing at least 250 microliters of deionized waterthrough the devices. After completion of the cyclization reaction thatintroduces the modified surface, the thickness of the layer increasedfrom 1.4 nm (functionalized surface thickness) to approx. 5 nm inthickness. Additionally, the sessile drop water contact angle decreasedfrom approximately 80 degrees (functionalized surface of Formula XV) to42 degrees (surface of Formula XXI).

Example 12. Introduction of a Propiolic Acid Modified Surface (FormulaXXII) to a Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was reacted with propiolic acid(HC≡CCO2H, Sigma Aldrich, Cat. # P51400-5G) by flowing at least 250microliters of a buffered saline solution (5.4×PBS pH 7.4) containing1.33 millimolar propiolic acid, 500 micromolar copper sulfate, 550micromolar THPTA ligand and 5 millimolar sodium ascorbate through themicrofluidic devices having the 11-azidoundecylsiloxy surface modifyingligand. The reaction was allowed to proceed at room temperature for atleast 1 hour or 40° C. for at least 15 minutes. The microfluidic devicehaving a carboxylic acid modified surface of Formula XXII was thenrinsed by flowing at least 250 microliters of deionized water throughthe devices. After completion of the cyclization reaction thatintroduces the modified surface, the thickness of the layer increasedfrom 1.1 nm (functionalized surface thickness) to 2 nm in thickness.Additionally, the sessile drop water contact angle decreased fromapproximately 80 degrees (functionalized surface of Formula XV) to 64degrees (surface of Formula XXII).

Example 13. Introduction of an Amine Modified Surface (Formula XXIII) toa Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was reacted with propargyl amine(HC≡CCH₂NH₂, Sigma Aldrich, Cat. # P50900-5G) by flowing at least 250microliters of a buffered saline solution (5.4×PBS pH 7.4) containing1.33 millimolar propargylamine, 500 micromolar copper sulfate, 550micromolar THPTA ligand and 5 millimolar sodium ascorbate through themicrofluidic devices having the 11-azidoundecylsiloxy surface modifyingligand. The reaction was allowed to proceed at room temperature for atleast 1 hour or 40° C. for at least 15 minutes. The microfluidic devicehaving a amine modified surface of Formula XXIII was then rinsed byflowing at least 250 microliters of deionized water through the devices.

Example 14. Introduction of a PEG Carboxylic Acid Modified Surface(Formula XXIV) to a Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was reacted with Alkyne PEG acid (PEG(f=5000 Da, Compound 11) Nanocs, Cat. # PG2-AKCA-5k) by flowing at least250 microliters of a buffered saline solution (5.4×PBS pH 7.4)containing 1.33 millimolar Compound 11, 500 micromolar copper sulfate,550 micromolar THPTA ligand and 5 millimolar sodium ascorbate throughthe microfluidic devices having the 11-azidoundecylsiloxy surfacemodifying ligand. The reaction was allowed to proceed at roomtemperature for at least 1 hour or 40° C. for at least 15 minutes. Themicrofluidic device having a carboxylic acid modified surface of FormulaXXIV was then rinsed by flowing at least 250 microliters of deionizedwater through the devices. After completion of the cyclization reactionthat introduces the modified surface, the thickness of the layerincreased from 1.1 nm (functionalized surface thickness) to 5 nm inthickness. Additionally, the sessile drop water contact angle decreasedfrom approximately 80 degrees (functionalized surface of Formula XV) to48 degrees (surface of Formula XXIV).

Example 15. Introduction of a Poly Lysine Modified Surface (Formula XXV)to a Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was reacted with poly(lysinehydrobromide)graft -(4 pentynamide, Compound 12, PLKB100-g-AK20AlamandaPolymers, Cat. # PLKB100-g-AK20, 100 lysine repeat units, 20%alkynylated, MW 21,000 Da) by flowing at least 250 microliters of abuffered saline solution (5.4×PBS pH 7.4) containing 1.33 millimolarCompound 12, 500 micromolar copper sulfate, 550 micromolar THPTA ligandand 5 millimolar sodium ascorbate through the microfluidic deviceshaving the 11-azidoundecylsiloxy surface modifying ligand. The reactionwas allowed to proceed at room temperature for at least 1 hour or 40° C.for at least 15 minutes. The microfluidic device having an aminemodified surface of Formula XXV was then rinsed by flowing at least 250microliters of deionized water through the devices. After completion ofthe cyclization reaction that introduces the modified surface, thethickness of the layer increased from 1.1 nm (functionalized surfacethickness) to approx. 3 nm in thickness. Additionally, the sessile dropwater contact angle decreased from approximately 80 degrees(functionalized surface of Formula XV) to 50 degrees (surface of FormulaXXV).

Example 16. Introduction of a Poly Glutamic Acid Modified Surface(Formula XXVI) to a Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was reacted with poly(glutamicacid)graft —(N-propargyl), Compound 13, Alamanda Polymers, Cat. #PLE100-g-AK20, 20% alkynylated, 100 glutamic acid repeats, MW 15,000 Da)by flowing at least 250 microliters of a buffered saline solution(5.4×PBS pH 7.4) containing 1.33 millimolar Compound 13, 500 micromolarcopper sulfate, 550 micromolar THPTA ligand and 5 millimolar sodiumascorbate through the microfluidic devices having the11-azidoundecylsiloxy surface modifying ligand. The reaction was allowedto proceed at room temperature for at least 1 hour or 40° C. for atleast 15 minutes. The microfluidic device having a carboxylic acidmodified surface of Formula XXVI was then rinsed by flowing at least 250microliters of deionized water through the devices. After completion ofthe cyclization reaction that introduces the modified surface, thethickness of the layer increased from 1.1 nm (functionalized surfacethickness) to approx. 3 nm in thickness. Additionally, the sessile dropwater contact angle decreased from approximately 80 degrees(functionalized surface of Formula XV) to 54 degrees (surface of FormulaXXVI).

Example 17. Introduction of a Biotinylated Polyethylene Glycol (PEG)Modified Surface with a Disulfide Cleavable Linker (Formula XXVII) to aMicrofluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV as described above, was treated with dibenzylcyclooctynyl(DBCO)S—S biotin modified-PEG3, Compound 14, Broadpharm, Cat. #BP-22453) by flowing at least 250 microliters of an aqueous solutioncontaining 1.33 micromolar Compound 14 through the microfluidic devicehaving surface modifying azide ligands after vapor deposition. Thereaction was allowed to proceed at 40° C. for at least 1 h. Themicrofluidic device having a modified surface of Formula XXVII was thenrinsed by flowing at least 250 microliters of DI water through thechips. One of two regioisomers shown.

After completion of the cyclization reaction that introduces themodified surface, the thickness of the layer increased from 1.1 nm(functionalized surface thickness) to approx. 2 nm in thickness.Additionally, the sessile drop water contact angle decreased fromapproximately 80 degrees (functionalized surface of Formula XV) to 66degrees (surface of Formula XXVII).

Example 18. Introduction of a PEG5 Carboxylic Acid Modified Surface(Formula XXVIII) to a Microfluidic Device

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was treated with dibenzylcyclooctynyl(DBCO)-PEGS-acid, Compound 15, Broadpharm, Cat. # BP-22449) by flowingat least 250 microliters of an aqueous solution containing 1.33micromolar Compound 15 through the microfluidic device having surfacemodifying azide ligands after vapor deposition. The reaction was allowedto proceed at 40° C. for at least 1 h. The microfluidic device having amodified surface of Formula XXVII was then rinsed by flowing at least250 microliters of DI water through the chips. The contact angle wasmeasured at 47°, and the thickness was 17.8 angstroms, each of which wasmeasured as described herein. One of two regioisomers shown.

Example 19. Introduction of a PEG3 Modified Surface (Formula XXIX) to aMicrofluidic Device

A microfluidic device (Berkeley Lights, Inc.) having a first siliconelectrode activation substrate and a second ITO substrate on theopposite wall, and photopatterned silicone microfluidic circuit materialseparating the two substrates, was treated in an oxygen plasma cleaner(Nordson Asymtek) for 1 min, using 100 W power, 240 mTorr pressure and440 sccm oxygen flow rate. The plasma treated microfluidic device wastreated in a vacuum reactor with methoxytriethyleneoxypropyltrimethoxysilane (Compound 16, Gelest Catalog # SIM6493.4, 300microliters) in a foil boat in the bottom of the vacuum reactor in thepresence of magnesium sulfate heptahydrate (0.5 g, Acros), as a waterreactant source in a separate foil boat in the bottom of the vacuumreactor. The chamber was then pumped to 750 mTorr using a vacuum pumpand then sealed. The vacuum reactor was placed within an oven heated at110° C. for 24-48 h. This introduced a functionalized surface to all ofthe inner facing surfaces of the microfluidic device, where thefunctionalized surface had a structure of Formula XXIX:

where Z is a bond to an adjacent silicon atom bound to the surface or isa bond to the surface and

is the surface. After cooling to room temperature and introducing argonto the evacuated chamber, the microfluidic device was removed from thereactor. The contact angle for this surface was measured to be 55° andthe average thickness to be 10.2 angstroms.

Example 20. Preparation of a Phosphonate Linked Surface (Formula XXXVI)

A silicon chip (780 microns thick, 1 cm by 1 cm) was pretreated asdescribed above for Example 3, and subsequently treated withoctadecylphosphonic acid (Compound 17, Sigma Aldrich Cat. #715166) as inExample 19 to provide the covalently modified surface of Formula XXXVI,where Z is a bond to a phosphorus atom in an adjacent linking group LGor is a bond to the surface

. The contact angle was measured to be 110°.

Example 21. Introduction of a Streptavidin Modified Surface (FormulaXXXVII or Formula XXXVIII) to a Microfluidic Device. Method A

The product microfluidic device from Example 5, having a surface ofFormula XV as described above, was treated with dibenzylcyclooctynyl(DBCO) Streptavidin (SAV) Compound 18, Nanocs, Cat. # SV1-DB-1, wherethere are 2-7 DBCO for each molecule of SAV) by flowing at least 250microliters of an aqueous solution containing 2 micromolar Compound 18through the microfluidic device having surface modifying azide ligandsafter vapor deposition. The reaction was allowed to proceed at roomtemperature for at least 1 h. The microfluidic device having a modifiedsurface of Formula XXVII was then rinsed by flowing at least 250microliters of 1×PBS through the device.

Method B.

The product modified surface of a microfluidic device of Example 17,having a surface of Formula XXVII, was washed with water and dried withrepeated flushes of gaseous carbon dioxide while heating the chip to 40°C. A solution of 2 micromolar SAV in 1×PBS (ThermoFisher catalog#434301) was flowed into the microfluidic device and contacted with thebiotinylated surface for 30 min. The excess SAV was removed by flowing1×PBS through the microfluidic device, providing a surface of FormulaXXXVIII:

Example 22. Introduction of a Fibronectin Surface (Formula XXXIX) withina Microfluidic Device. Method A

The product of Example 21, method B, a microfluidic device having amodified surface of Formula XXXVIII was treated with 50 microliters of asolution of 46 nM biotinylated bovine fibronectin (randomlybiotinylated, Cytoskeleton Inc., Catalog #FNR03A, FNR03-B) in 1×PBS,which was incubated for one hour at 37° C., providing the surface offibronectin of Formula XXXXIX:

Modification in the Presence of Biological Cells.

In some embodiments, biological cells were introduced to themicrofluidic device having surfaces of Formula XXXVIII, presentingstreptavidin to the fluidic regions of the microfluidic device. Afterthe cells were imported into sequestration pens, biotinylatedfibronectin was introduced in PBS, and incubated for 1 hour. Adherencewas observed.

Method B.

A fibronectin surface is introduced by treating a microfluidic devicehaving a surface of Formula XXXVII as above with biotinylatedfibronectin.

Method C.

A biotinylated-fibronectin stock was prepared at 2.3 micromolar in PBSand streptavidin stock was prepared at 19.2 micromolar in PBS. The twowere mixed at fibronectin to streptavidin ratios of 1:1 to 1:2 anddiluted in IX PBS to final concentration of at least 300 nanomolar. Thissolution was incubated for 15 minutes at room temperature to allowcoupling of the fibronectin and streptavidin, forming a surfacemodifying reagent having a coupling group CG of biotin/streptavidin.

The product modified surface of a microfluidic device of Example 17,having a surface of Formula XXVII, was washed with water and dried withrepeated flushes of gaseous carbon dioxide while heating the chip to 40°C. The pre-formed surface modifying reagent of SAV bound to biotinylatedfibronectin was above was flowed into the microfluidic device. Thedevice was incubated at room temperature for at least 30 minutes, andprovided a modified surface of Formula XXXIX.

Further Generalization.

Additionally, any number of biologically relevant molecules may beintroduced into a modified surface of a microfluidic device by the sameprocess, by flowing in a biotinylated protein, peptide, small moleculeor recognition motif, by attachment to either a surface of FormulaXXXVII or Formula XXXVIII. For example, biotinylated laminin is flowedinto a microfluidic device prepared as above with a surface of FormulaXXXVII or XXXVIII, to produce a modified surface having laminin surfacecontact moieties (Formula XL):

Example 23. Introduction of a Mixed Surface of Formula XLI in VaryingRatios

A silicon wafer was treated in an oxygen plasma cleaner (NordsonAsymtek) for 1 min, using 100 W power, 240 mTorr pressure and 440 sccmoxygen flow rate. The plasma treated microfluidic device was treated ina vacuum reactor with mixture of 3-azidoundecyl) trimethoxy silane(prepared as described above, Compound 5) andmethoxytriethyleneoxypropyltrimethoxy silane (Gelest Inc. Catalog #SIM6493.4, having a similar molecular weight as Compound 5, in varyingratios) in a foil boat in the bottom of the vacuum reactor in thepresence of magnesium sulfate heptahydrate (0.5 g, Acros), as a waterreactant source in a separate foil boat in the bottom of the vacuumreactor. The chamber was then pumped to 750 mTorr using a vacuum pumpand then sealed. The vacuum reactor was placed within an oven heated at110° C. for 24-48 h.

After cooling to room temperature and introducing argon to the evacuatedchamber, the wafer was removed from the reactor. The wafer was rinsedwith acetone, isopropanol, and dried under a stream of nitrogen. Themodified surface of Formula XLI, which was a mixture where x and y maybe present in a ratio of x:y or y:x of any value from 1 to 1×10⁸, wasevaluated for thickness, contact angle and for the presence of azide inthe FTIR of the surface. Individual wafers were modified with mixturesof 1% undecyl azide: 99% methoxy PEG3; 10% undecyl azide: 90% methoxyPEG3; 50% undecyl azide: 50% methoxy PEG3: and 100% undecyl azide.

As shown in FIG. 5A, the overlaid FTIR traces clearly showed diminishingamount of azide asymmetric stretch 510 at ˜2098 cm-1. FIG. 5B shows anenlarged portion of the overlaid traces at the location of the azideasymmetric stretch for the wafers having 10% Formula XV, and 1% FormulaXV respectively. The relative amounts of azide were clearlydistinguishable and correlated to the ratios of Formula XV: Formula XXIXused.

The contact angle and thickness of the modified surface also differedwhen differing ratios of surfaces of Formula XV: Formula XXIX werepresent on the modified surface, as shown in Table 2: The data showsthat control of deposition was obtained by changing the ratio ofmaterials during the chemical vapor deposition process. The change incontact angle also shows that differing performance was possible withdiffering ratios of these surface modifications.

TABLE 2 Physical measurement of mixed surfaces. % Thickness ContactAzide (Å) Angle  1% 13.76 56°  10% 12.28 62°  50% 9.35 68° 100% 13.3185°

Example 24. Introduction of a Modified Surface Having a Mixture of aFirst Surface Modification Containing PEG and a Second SurfaceModification of a Block Copolymer Containing Poly-L-Lysine, (FormulaXLII) Using a Combination of Surface Modifying Reagents

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was treated with a solution including1.3 millimolar propargyl-PEG1-disulfide-PEG1-propargyl (Compound 19,BroadPharm Inc. Catalog # BP-23283), with copper sulfate (in excess),THPTA ligand, and sodium ascorbate. The excess copper sulfate preventsdisulfide cleavage by ascorbate during the reaction, which was performedat 40° C. for about 15 min (and may alternatively be performed for about1 hr at room temperature). After the incubation period was complete, theexcess reagent and byproducts were removed by flushing with water. Theinterior of the microfluidic device was dried by flushing with carbondioxide gas while heating the microfluidic device to 40° C., providing asurface that is a secondary functionalized surface having alkynyl R_(x2)moieties.

The microfluidic device having secondary functionalized surfaces havingalkynyl R_(x2) moieties was then further modified by treating themicrofluidic device with a mixture of two surface modifying reagents.The surface modifying reagents were flowed into the microfluidic deviceat a 1.3 millimolar concentration of a mixture of PEG-Azide (5 Kda,Aldrich Chemicals, Catalog #689475): azide-PEG5k-block copolymerpoly-1-lysine 100 (Alamanda Polymers, MW 1600), where the ratio ofazide-PEG and azide-PEG5k-b-PLL was varied between 1:50000 to 50000:1;along with copper sulfate (in excess), THPTA ligand, and sodiumascorbate. The excess copper sulfate prevents disulfide cleavage byascorbate during the reaction, which was performed at 40° C. for about15 min (and may alternatively be performed for about 1 hr at roomtemperature). After the incubation period was complete, the excessreagent and byproducts were removed by flushing with water. The interiorof the microfluidic device was dried by flushing with carbon dioxide gaswhile heating the microfluidic device to 40° C., providing amicrofluidic device with a mixture of a first surface modification,PEG5K, that is hydrophilic and a second surface modification,PEG5k-b-PLL, where the block of PLL provided positive charge (FormulaXLII). The proportion of azide-PEG and azide-PEG5k-b-PLL may be an evenhigher ratio, e.g., 10,000:1 or more, as it was demonstrated thatadhesion is observed even at extremely low proportions of the blockcopolymer poly-L-lysine surface contact moiety.

The modified surface of Formula XLII, may have x and y present in aratio of x:y or y:x of any value from 1 to 1×10⁸.

Alternative Method of Modification.

The microfluidic device having a surface of Formula XV may be modifiedwith DBCO-PEG4-alkyne (Compound 20, Conju-Probes, Inc. Catalog #CP-2039), in place of propargyl-PEG1-disulfide-PEG1-propargyl (Compound19). by flowing at least 250 microliters of an aqueous solutioncontaining 1.0 millimolar DBCO-PEG through the microfluidic devicehaving surfaces of Formula XV. The reaction was allowed to proceed at40° C. for at least 1 h. The microfluidic device having a modifiedsurface of Formula XVIII was then rinsed by flowing at least 250microliters of DI water through the chips, and may be treated asdescribed in the preceding paragraphs to provide a microfluidic devicewith a mixture of a first surface modification, PEG5K, that ishydrophilic and a second surface modification, PEG5k-b-PLL, where theblock of PLL provides positive charge (Formula XLIII), where the linkerportion of the surface modifications differ from that of Formula XLII.

The modified surface of Formula XLIII, may have x and y present in aratio of x:y or y:x of any value from 1 to 1×10⁸.

Useful for Culturing Adherent Cells.

Surfaces of either Formula XLII or XLIII were useful in providinganchoring points (e.g., clusters of positively charged poly-L-lysineprovided within the block co-polymer) for culturing adherent cells suchas, but not limited to, HeLa cells. HeLa cells were observed to flattenout, grow and multiply during culture on either of these surfaces (datanot shown).

Example 25. Introduction of a Modified Surface Having a Mixture of aFirst Surface Modification Containing PEG and a Second SurfaceModification of a Poly-L-Lysine, (Formula XLIV) Using a Combination ofSurface Modifying Reagents

The product microfluidic device from Example 5, having a surface ofFormula XV, as described above, was treated with a 1.33 millimolarsolution including a 1:1 stoichiometric mixture of alkyne-poly-L-lysineHBr salt (100mer unit, Alamanda Polymers) and alkyne-modified PEG (j=MW˜5000 Da, Compound 6, JenKem Technologies) along with copper sulfate (inexcess), THPTA ligand, and sodium ascorbate. The excess copper sulfateprevents disulfide cleavage by ascorbate during the reaction, which wasperformed at 40° C. for about 15 min (and may alternatively be performedfor about 1 hr at room temperature). After the incubation period wascomplete, the excess reagent and byproducts were removed by flushingwith water. The interior of the microfluidic device was dried byflushing with carbon dioxide gas while heating the microfluidic deviceto 40° C., providing a microfluidic device with a mixture of a firstsurface modification, PEG5K, that is hydrophilic and a second surfacemodification, poly-L-lysine, which provides positive charge (FormulaXLIV).

In the surfaces of Formula XLIV, (*) are proprietary linkers, and x andy may be present in a ratio of x:y or y:x of any value from 1 to 1×10⁸.

Useful for Culturing Adherent Cells.

Surfaces of Formula XLIV were useful in providing anchoring points(e.g., clusters of positively charged poly-L-lysine provided within theblock co-polymer) for culturing adherent cells such as, but not limitedto, HeLa cells. HeLa cells were observed to flatten out, grow andmultiply during culture on either of these surfaces (data not shown).

Titration of Surface Modification 1: Surface Modification 2.

The ratios of the first surface modification (PEG 5 kDa) and the secondsurface modification (poly-L-lysine) of Formula XLIV were modified tomodulate the population of points designed to encourage adherence, downto a ratio of 99 PEG 5 kDa: 1 poly-L-lysine. Using a 1% level of chargedsecond surface modification (poly-L-lysine) laser bubble initiation ofdisplacement, followed by export by dielectrophoretic force of cells wasseen (Data not shown).

A microfluidic device having inner surfaces modified with a firstsurface modification of PEG 5 kDa and a second surface modification ofpoly-L-lysine having a percentage of poly-L-lysine surface modificationsof about 0.00001% or 0.000001% is expected to permit adhesion ofadherent cells (such as HeLa cells) while still permitting export ofcultured cells using dielectrophoretic forces, without laser initiationof displacement.

Example 26. Introduction of a Mixed Surface Using Branched PEG Linkers(Formula XLV)

A modified surface having a first cleavable biotinylated surfacemodification in combination with a second surface modification which washydrophilic PEG was introduced using a multi-armed PEG alkyne moiety.The amount of biotin reactive moieties present was controlled bymodulating the amount of biotin reactive moiety vs hydrophilic surfacecontact moiety. Modulation was achieved by coupling moieties containingeach of the surface contact moieties to arms of a multiarmed PEG alkyne,leaving sufficient alkyne reactive moieties on the multiarm PEG toeffect efficient modification of the surface of the microfluidic device.The following procedure is described for a 1:1 ratio of biotin moietiesto PEG carboxylic acid moieties, but experiments were also conducted for100% biotin moieties; 10% biotin to 90% PEG carboxylic acid; and 1%biotin moieties to 99% PEG carboxylic acid moieties.

A solution of 1.3 millimolar 4-arm PEG (Creative PEGWorks Catalog#PSB-495), 1.3 millimolar solution of a 1:1 ratio ofazide-disulfide-biotin (Compound 20, BroadPharm Catalog # BP-22877) andazide-PEG6-carboxylic acid (BroadPharm Catalog # BP-20612) in aqueoussolution was reacted with sodium ascorbate, in the presence of a twofoldexcess of copper sulfate to form bi-modified multi-arm PEG uponincubation at room temperature for about 30 min. The solution ofbi-modified multi-arm PEG was introduced onto a silicon wafer fromExample 3, having a surface of Formula XV, as described above, with anadditional aliquot of sodium ascorbate with a twofold excess of coppersulfate. The remaining alkyne ligands of the multi-arm PEG reacted withthe azide reactive moieties of the surface of Formula XV to produce amixed modified surface having a reactive moiety of biotin and a surfacecontact moiety of PEG-carboxylic acid (Formula XLV).

This mixed surface was then further modified by addition of 1 micromolarsolution of Streptavidin (SAV) in PBS and incubated for 15 min at roomtemperature, producing a surface of Formula XLVI, where a first surfacecontact moiety is PEG-COOH and a second reactive moiety is SAV. Thesample was washed and the thickness of the modified surface wasmeasured.

The thicknesses of the modified layers are shown in Table 3, and vary asexpected with the varying amount of streptavidin bound to availablebiotin surface contact moieties

TABLE 3 Measured thickness of modified surfaces. Total Total Increase ofModified Modified thickness surface surface due to having having addedmulti-arm multi-arm multi-arm Surface of PEG with PEG with PEG withFormula XV biotin and biotin/SAV biotin/SAV (base PEG and PEG and PEGsurface) in COOH in COOH in COOH in Sample angstroms angstroms angstromsangstroms 100% biotin 11.8 33.7 60.6 26.9 50% biotin:50% 11.8 31.8 51.619.8 PEG COOH 10% biotin:90% 12.0 30.8 44.2 13.4 PEG COOH 1% biotin:99%12.2 30.8 34.3  3.4 PEG COOH

The results show that a modulated surface having a combination of astreptavidin reactive moiety and a PEG COOH surface contact moiety wasobtained. The streptavidin cam be modified further with biotinylatedspecies such as biotin-fibronectin or any moiety capable of beingbiotinylated, to obtain a mixed surface of a first contact moiety (e.g.,fibronectin) and a second contact moiety of PEG COOH, in any desiredratio.

Example 27. Introduction of Regioselective Surface Modifications ofPEG5k in a First Region of the Microfluidic Device and Poly-L-Lysinewithin Sequestration Pens (Formula XLVII)

A previously prepared, dry and unprimed (e.g., not flushed with carbondioxide gas) microfluidic device having a surface of Formula XV wastreated with a 1.0 to 3.3 milllimolar aqueous solution ofdibenzylcyclooctynyl (DBCO) modified-PEG, weight averaged molecularweight 5000 Da (Compound 8, Broadpharm, Cat. # BP-22461) by aspiratingthe solution through the microfluidic channel of the device at slightlylower than atmospheric pressure. The channel was resultingly filled withthe reagent solution. However, due to the low pressure of the fluidicintroduction and the unprimed nature of the surfaces within themicrofluidic device, the DBCO modified PEG5 kDa solution does not enterthe sequestration pens opening off of the microfluidic channel. Afterincubation for 30 min at room temperature, 80 microliters of water wasaspirated at reduced pressure through the channel, washing any remainingreagent out of the microfluidic device. The solution was stillcontrolled to flow only through the microfluidic channel. Additionalflushing with water at 1 microliter/sec at low pressure was continuedfor about 5 min. The surface modified microfluidic channel was flushedwith carbon dioxide gas repeatedly, while heating the device to 90° C.

The dried microfluidic device having a first surface modification ofPEG5K was then primed with carbon dioxide as described above. Thesequestration pens opening off of the microfluidic channel then weremodified regioselectively by flowing in a 1.33 micromolar solutionincluding a 1:1 stoichiometric mixture of alkyne-poly-L-lysine HBr salt(100mer unit, Alamanda Polymers) and alkyne-modified PEG (j=MW ˜5000 Da,Compound 6, JenKem Technologies) along with copper sulfate (in excess),THPTA ligand, and sodium ascorbate. The excess copper sulfate preventsdisulfide cleavage by ascorbate during the reaction, which was performedat 40° C. for about 15 min (and may alternatively be performed for about1 hr at room temperature). After the incubation period was complete, theexcess reagent and byproducts were removed by flushing with water. Theinterior of the microfluidic device was dried by flushing with carbondioxide gas while heating the microfluidic device to 40° C., providing amicrofluidic device with a regioselective introduction of a firstsurface modification, having only PEG5K, within the microfluidic channeland a second regioselective surface modification includingpoly-L-lysine, which provides positive charge for enhancing adherence ofbiological cells, only in the sequestration pens. (Formula XLV). Of noteis the ability to modulate the ratios of the reagents used to modify thesurface of the sequestration pens. The ratio of PEG-5K: poly-L-lysinewas varied from 0:100 to 99.9999:0.0001% and adherence of HeLa cells wasobserved within the sequestration pens, while migration of the motileHeLa cells was inhibited by the presence of the merely hydrophilicsurfaces within the channel.

A microfluidic device having inner surfaces modified with a firstsurface modification of PEG 5 kDa and a second surface modification ofpoly-L-lysine having a percentage of poly-L-lysine surface modificationsof about 0.00001% or 0.000001% is expected to permit adhesion ofadherent cells (such as HeLa cells) while still permitting export ofcultured cells using dielectrophoretic forces, without laser initiationof displacement.

Further Generalization.

Any type of surface modifying reagent may be used in introduction of thesecond surface modification within the sequestration pen, and is notlimited to a poly-L-lysine.

Secondary passivation of the microfluidic channel with a second surfacemodification only in the channel region. After the initial surfacemodification of the microfluidic channel as described above, there canbe unreacted reactive moieties (e.g., azide) still present in thechannel. Without wishing to be bound by theory, this may occur if themodifying reagent is bulky. Secondary passivation with a less stericallydemanding surface modifying reagent may be able to access remainingreactive moieties to add a second surface modification to the modifiedsurfaces of the channel without modifying the surfaces in thesequestration pen.

The microfluidic device, having a PEG5 kDa surface modificationintroduced to only to the microfluidic channel, was only rinsed withwater after the surface introduction. A second treatment withDBCO-PEG4-OH (Aldrich Catalog #761982 at a concentration of 1.3micromolar in an aqueous solution was performed similarly to the firsttreatment as described above. Since the microfluidic device was notprimed, none of the second surface modifying reagent entered thesequestration pens and accordingly only the channel was furthermodified. After washing, drying and heating, followed by carbon dioxidepriming, regioselective modification of the sequestration pens is thenperformed as above.

Example 28. Culturing of OKT3 Cells within a Microfluidic Device Havinga PEG Modified Surface

Materials:

OKT3 cells, a mouse B lymphocyte hybridoma, were obtained from theAmerican Type Culture Collection (ATCC) (catalog ATCC® CRL-8001™), andwere provided as a suspension cell line. Cultures were maintained byseeding 2×10⁵ viable cells/mL and incubating at 37° C., using 5% carbondioxide gaseous environment. Cells were split at 2×10⁴ cells/mL or 1×10⁵cells/mL every 2-3 days. Cells were frozen in 5% dimethyl sulfoxide(DMSO)/95% complete growth medium.

Culture Medium:

IMDM (Gibco, catalog 12440053) was supplemented with 20% Fetal BovineSerum (FBS) and 1% Penicillin-Streptomycin (10,000 U/mL) (Gibco, catalog15140122). Complete media was then filtered through a 0.2 μm PES,sterile membrane filter unit (Nalgene, 567-0020).

Priming and Perfusion Procedures:

As above, in general experimental detail section.

System and Microfluidic Device:

As above, in the general experimental detail section The sequestrationpens have a volume of about 7×10⁵ cubic microns.

Modified Microfluidic Surface.

The microfluidic device had a covalently linked PEG modified surface,prepared as described above in Example 6 (Formula XVI).

An OKT3 cell suspension in the culture medium was introduced into themicrofluidic device by flowing the suspension through a fluidic inletand into the microfluidic channel. The flow was stopped and the cellswere randomly loaded into sequestration pens by tilting the chip andallowing gravity to pull the cells into the sequestration pens.

After loading the OKT3 cells into the sequestration pens, the culturemedium was perfused through the microfluidic channel of the nanofluidicchip for a period of 3 days. FIG. 6A showed the growth of OKT3 cells onthe PEG-modified surface of the sequestration pens of the microfluidicdevice. The growth of OKT3 cells on the PEG surface was improvedrelative to a non-modified surface of a similar microfluidic device(data not shown).

The OKT3 cells were then removed from the sequestration pens by OET.FIG. 6B showed the extent of removal from the sequestration pen at theend of a twenty minute period, demonstrating excellent ability to exportthe expanded OKT3 cells into the flow channel, which was improved overthat of removal of OKT3 cells from a non-conditioned surface of asimilar microfluidic device. The OKT3 cells were then exported from themicrofluidic device (not shown).

Example 29: Culturing and Export of T Lymphocytes on a Dextran ModifiedMicrofluidic Surface

Materials. CD3+ cells from AllCells Inc. and mixed withanti-CD3/anti-CD28 magnetic beads (Dynabeads®, Thermofisher Scientific,Cat. No. 11453D) at a ratio of 1 bead/1 cell. The mixture was incubatedin the same medium as the culturing experiment itself, for 5 hours in a5% CO₂ incubator at 37° C. Following the incubation, the T cell/beadmixture was resuspended for use.

Culture Medium.

RPMI-1640 (GIBCO®, ThermoFisher Scientific, Cat. No. 11875-127), 10%FBS, 2% Human AB serum (50 U/ml IL2; R&D Systems).

Priming Procedure:

As above, in the general experimental detail section.

Perfusion Regime:

As above, in the general experimental detail section.

System and Microfluidic Device:

As above, in the general experimental detail section. The sequestrationpens have a volume of about 7×10⁵ cubic microns.

Modified Microfluidic Surface.

The microfluidic device had a covalently linked dextran modifiedsurface, prepared as described above in Example 7.

The T cell (plus bead) suspension was introduced into the microfluidicdevice by flowing the resuspension through a fluidic inlet and into themicrofluidic channel. The flow was stopped and T cells/beads wererandomly loaded into sequestration pens by tilting the chip and allowinggravity to pull the T cells/beads into the growth chambers.

After loading the T cells/beads into the sequestration pens, the culturemedium was perfused through the microfluidic channel of the nanofluidicchip for a period of 4 days. FIG. 7A showed the growth of T cells on thedextran modified surface of the sequestration pens of the microfluidicdevice. The growth of T cell on the dextran surface was improvedrelative to a non-conditioned surface of a similar microfluidic device(data not shown).

The T cells were then removed from the sequestration pens by gravity(e.g., tilting the microfluidic device). FIG. 7B showed the extent ofremoval from the sequestration pen at the end of a twenty minute period,demonstrating excellent ability to export the expanded T cells into theflow channel, which was improved over that of removal of T cells from anon-modified surface of a similar microfluidic device (data not shown).The T cells were then exported from the microfluidic device (not shown).

In addition to any previously indicated modification, numerous othervariations and alternative arrangements may be devised by those skilledin the art without departing from the spirit and scope of thisdescription. Thus, while the information has been described above withparticularity and detail in connection with what is presently deemed tobe the most practical and preferred aspects, it will be apparent tothose of ordinary skill in the art that numerous modifications,including, but not limited to, form, function, manner of operation, anduse may be made without departing from the principles and concepts setforth herein. As used herein, the examples and embodiments, in allrespects, are meant to be illustrative only and should not be construedto be limiting in any manner. It should also be noted, that while theterm step is used herein, that term may be used to simply draw attentionto different portions of the described methods and is not meant todelineate a starting point or a stopping point for any portion of themethods, or to be limiting in any other way.

Recitation of Some Embodiments of the Disclosure

1. A microfluidic device including: an enclosure including a base, acover, and microfluidic circuit material defining a fluidic circuittherein, where at least one inner surface of the base, the cover and themicrofluidic circuit material has a plurality of first covalently boundsurface modifications, each including a first linking group and a firstmoiety, where the first moiety is a first surface contact moiety or afirst reactive moiety; where at least one inner surface of the base, thecover and the microfluidic circuit material has a plurality of secondcovalently bound surface modifications, each including a second linkinggroup and a second moiety, where the second moiety is a second surfacecontact moiety or second reactive moiety, and where the first linkinggroup and the second linking group are different from each other and/orthe first moiety is different from the second moiety.

2. The microfluidic device of embodiment 1, where the first moiety andthe second moiety may each be covalently bound to the surface via alinking group LG independently selected from —W—Si(OZ)₂O— and —OP(O)₂O—,where W is O, S, or N, and where Z is a bond to a silicon atom in anadjacent linking group LG or is a bond to the surface.

3. The microfluidic device of embodiment 1 or 2, where the first surfacecontact moiety may include one or more of an alkyl, fluoroalkyl,monosaccharide, polysaccharide, alcohol, polyalcohol, alkylene ether,polyelectrolytes, amino, carboxylic acid, phosphonic acid, sulfonateanion, carboxybetaines, sulfobetaine, sulfamic acid, amino acid moiety,or cleavable moiety; and/or where the second surface contact moiety mayinclude one or more of an alkyl, fluoroalkyl, monosaccharide,polysaccharide, alcohol, polyalcohol, alkylene ether, polyelectrolytes,amino, carboxylic acid, phosphonic acid, sulfonate anion,carboxybetaines, sulfobetaine, sulfamic acid, amino acid moiety, orcleavable moiety.

4. The microfluidic device of embodiment 1 or 2, where the first surfacecontact moiety may include a polyethylene glycol moiety, a dextranmoiety, a proteinaceous moiety, a poly carboxylic acid, a polylysinemoiety, or any combination thereof; and/or where the second surfacecontact moiety may include a polyethylene glycol moiety, a dextranmoiety, a proteinaceous moiety, a poly carboxylic acid, a polylysinemoiety, or any combination thereof.

5. The microfluidic device of any one of embodiments 1 to 4, where thefirst reactive moiety may be an alkyne moiety, an azide moiety, acarboxylic acid moiety, an amine moiety, an olefinic moiety, atetrazinyl moiety, a trans-cyclooctenyl moiety, a thiol moiety, amaleimide moiety, a biotin moiety, a streptavidin moiety, a halidemoiety, a cyano moiety, isocyanate moiety, an epoxide moiety, ahydroxyamine moiety, or a sulfonyl fluoride moiety; and/or where thesecond reactive moiety may be an alkyne moiety, an azide moiety, acarboxylic acid moiety, an amine moiety, an olefinic moiety, atetrazinyl moiety, a trans-cyclooctenyl moiety, a thiol moiety, amaleimide moiety, a biotin moiety, a streptavidin moiety, a halidemoiety, a cyano moiety, isocyanate moiety, an epoxide moiety, ahydroxyamine moiety, or a sulfonyl fluoride moiety.

6. The microfluidic device of any one of embodiments 1 to 5, where thefirst covalently bound surface modifications may include a linker, wherethe linker includes 1 to 200 non-hydrogen atoms selected from anycombination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorusatoms; and/or where the second covalently bound surface modificationsmay include a linker, where the linker may include 1 to 200 non-hydrogenatoms selected from any combination of silicon, carbon, nitrogen,oxygen, sulfur and phosphorus atoms.

7. The microfluidic device of embodiment 6, where the linker of thefirst covalently bound surface modifications may further include one ortwo coupling group CG moieties; and/or where the linker of the secondcovalently bound surface modifications may further include one or twocoupling group CG moieties.

8. The microfluidic device of embodiment 1, where the first covalentlybound surface modifications may have a structure selected from FormulaXXX, Formula V, Formula VII, Formula XXXI, Formula VIII, and Formula IX:

where: LG is —W—Si(OZ)₂O— or —OP(O)₂O—; L_(fm) is a linker including 1to 200 non-hydrogen atoms selected from any combination of silicon,carbon, nitrogen, oxygen, sulfur and phosphorus atoms and may furtherinclude 0 or 1 coupling groups CG; R_(x) is a reactive moiety; W is O,S, or N; Z is a bond to an adjacent silicon atom or is a bond to thesurface; n is an integer of 3 to 21; L_(sm) is a linker including 1 to200 non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms and further may include 0,1, 2, or 3 coupling groups CG; and

is the surface.

9. The microfluidic device of embodiment 8, where LG may be—W—Si(OZ)₂O—, and where W may be O.

10. The microfluidic device of embodiment 8 or 9, where n is 7 to 21.

11. The microfluidic device of any one of embodiments 8 to 10, where thereactive moiety R_(x) may be alkyne, azide, amine, carboxylic acid,biotin, streptavidin, olefin, trans cyclooctene, s-tetrazine, thiol,maleimide, halide, cyano, isocyanate, epoxide, hydroxyamine, a maskedhydroxyl, or sulfonyl fluoride.

12. The microfluidic device of any one of embodiments 8 to 10, where thereactive moiety R_(x) may be alkyne, azide, amine, carboxylic acid,biotin, or streptavidin.

13. The microfluidic device of any one of embodiments 1 or 8-12, wherethe second covalently bound surface modifications may have a structureselected from Formula XXX′, Formula V′, Formula VII′, Formula XXXI′,Formula VIII′, and Formula IX′:

where: LG′ is —W′—Si(OZ′)₂O— or —OP(O)₂O—; L′_(fm) is a linker including1 to 200 non-hydrogen atoms selected from any combination of silicon,carbon, nitrogen, oxygen, sulfur and phosphorus atoms and may furtherinclude 0 or 1 coupling groups CG; R_(x)′ is a reactive moiety; W′ is O,S, or N; Z′ is a bond to an adjacent silicon atom or is a bond to thesurface; n′ is an integer of 3 to 21; L′_(sm) is a linker including 1 to200 non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms and further may include 0,1, 2, or 3 coupling groups CG; and

is the surface.

14. The microfluidic device of embodiment 13, where LG′ may be—W′—Si(OZ′)₂O—, and where W′ may be O.

15. The microfluidic device of embodiment 13 or 14, where n′ may be 7 to21.

16. The microfluidic device of any one of embodiments 13 to 15, wherethe reactive moiety R_(x)′ may be alkyne, azide, amine, carboxylic acid,biotin, streptavidin, olefin, trans cyclooctene, s-tetrazine, thiol,maleimide, halide, cyano, isocyanate, epoxide, hydroxyamine, a maskedhydroxyl, or sulfonyl fluoride.

17. The microfluidic device of any one of embodiments 13 to 15, wherethe reactive moiety R_(x)′ may be alkyne, azide, amine, carboxylic acid,biotin, or streptavidin.

18. The microfluidic device of any one of embodiments 1 to 17, where thefirst moiety may be different from the second moiety.

19. The microfluidic device of any one of embodiments 13 to 17, wherethe first covalently bound surface modifications may have a structureselected from Formula XXX, Formula V, and Formula VII, and where thesecond covalently bound surface modifications may have a structureselected from Formula XXXI′, Formula VIII′, and Formula IX′.

20. The microfluidic device of embodiment 19, where the first covalentlybound surface modifications and the second covalently bound surfacemodifications may be on a common inner surface of the base, the cover,and/or the microfluidic circuit material.

21. The microfluidic device of embodiment 20, where the first and secondcovalently bound surface modifications may be randomly distributed uponthe common inner surface.

22. The microfluidic device of embodiment 20, where the common innersurface may include a first region including the first covalently boundsurface modifications and a second region including the secondcovalently bound surface modifications, where the first region isadjacent to the second region.

23. The microfluidic device of embodiment 20, where the common innersurface may include a plurality of first regions including the firstcovalently bound surface modifications and a second region including thesecond covalently bound surface modifications, where the first regionsof the plurality are separated from each other by or each adjacent tothe second region.

24. The microfluidic device of embodiment 20, where the common innersurface may include a plurality of second regions including the secondcovalently bound surface modifications and a first region including thefirst covalently bound surface modifications, where the second regionsof the plurality are separated from each other by or each adjacent tothe first region.

25. The microfluidic device of any one of embodiments 13 to 18, wherethe first covalently bound surface modifications may have a structureselected from Formula XXXI, Formula VIII, and Formula IX, where thesecond covalently bound surface modifications may have a structureselected from Formula XXXI′, Formula VIII′ and Formula IX′, and wherethe first covalently bound surface modifications are different from thesecond covalently bound surface modifications.

26. The microfluidic device of embodiment 25, where the surfacemodifying ligand of the first covalently bound surface modifications mayhave a structure of Formula X, and where the surface modifying ligand ofthe second covalently bound surface modifications may have a structureof Formula XI:

where: CG is a coupling group; and L is a linker including a bond or 1to 200 non-hydrogen atoms selected from any combination of silicon,carbon, nitrogen, oxygen, sulfur and phosphorus atoms.

27. The microfluidic device of embodiment 25 or 26, where the firstcovalently bound surface modifications and the second covalently boundsurface modifications may be on a common inner surface of the base, thecover, and/or the microfluidic circuit material.

28. The microfluidic device of embodiment 27, where the first and secondcovalently bound surface modifications may be randomly distributed uponthe common inner surface.

29. The microfluidic device of embodiment 27, where the common innersurface may have a first region including the first covalently boundsurface modifications and a second region including the secondcovalently bound surface modifications, and where the first region isadjacent to the second region.

30. The microfluidic device of embodiment 27, where the common innersurface may include a plurality of first regions having the firstcovalently bound surface modifications and a second region having thesecond covalently bound surface modifications, where the first regionsof the plurality are separated from each other by or each adjacent tothe second region.

31. The microfluidic device of any one of embodiments 27 to 30, wherethe common inner surface may include more than one kind of proteinaceousmoiety.

32. The microfluidic device of any one of embodiments 25 to 31, wherethe surface modifying ligand of the first covalently bound surfacemodifications may include a first proteinaceous moiety, and where thesurface modifying ligand of the second covalently bound surfacemodifications may include a second proteinaceous moiety, and where thefirst and second proteinaceous moieties are different.

33. The microfluidic device of any one of embodiments 13 to 18, wherethe first covalently bound surface modifications may have a structureselected from Formula XXX, Formula V, and Formula VII, where the secondcovalently bound surface modifications may have a structure selectedfrom Formula XXX′, Formula V′, and Formula VII′, where the firstcovalently bound surface modifications are different from the secondcovalently bound surface modifications, and where the reactive moiety ofthe first covalently bound surface modifications does not react with thereactive moiety of the second covalently bound surface modifications.

34. The microfluidic device of embodiment 33, where the first covalentlybound surface modifications and the second covalently bound surfacemodifications may be on a common inner surface of the base, the cover,and/or the microfluidic circuit material.

35. The microfluidic device of embodiment 34, where the common innersurface may include a first region including the first covalently boundsurface modifications and a second region including the secondcovalently bound surface modifications, and where the first region isadjacent to the second region.

36. The microfluidic device of embodiment 34, where the common innersurface may include a plurality of first regions including the firstcovalently bound surface modifications and a second region including thesecond covalently bound surface modifications, where the first regionsof the plurality are separated from each other by or each adjacent tothe second region.

37. The microfluidic device of any one of embodiments 1 to 36, where thefluidic circuit may include a flow region and a sequestration pen, wherethe sequestration pen may include an isolation region and a connectionregion, where the connection region may include a proximal opening tothe flow region and fluidically connects the isolation region to theflow region.

38. The microfluidic device of embodiment 37, where at least one surfaceof the flow region may be modified with the first covalently boundsurface modifications, where at least one surface of the sequestrationpen may be modified with the second covalently bound surfacemodifications.

39. The microfluidic device of embodiment 38, where the secondcovalently bound surface modifications may include a surface contactmoiety configured to anchor adherent cells.

40. The microfluidic device of embodiment 38 or 39, where the firstcovalently bound surface modifications may include a surface contactmoiety configured to inhibit or substantially prevent migration ofmotile cells out of the sequestration pen.

41. The microfluidic device of any one of embodiments 37 to 40, wherethe flow region may be fluidically connected to a fluidic inlet and afluidic outlet, and may be configured to contain a flow of a firstfluidic medium.

42. The microfluidic device of any one of embodiments 37 to 41, wherethe sequestration pen may include walls made of microfluidic circuitmaterial.

43. The microfluidic device of embodiment 42, where the walls of thesequestration pen may extend from the inner surface of the base to theinner surface of the cover.

44. The microfluidic device of any one of embodiments 37 to 43, wherethe inner surface of the base may underlay the flow region and aninterior of the sequestration pen.

45. The microfluidic device of any one of embodiments 37 to 44, wherethe fluidic circuit further may include a plurality of sequestrationpens each having at least one inner surface modified with the firstand/or second covalently bound surface modifications.

46. The microfluidic device of any one of embodiments 1 to 45, where thefirst covalently bound surface modifications and/or the secondcovalently bound surface modifications may form a monolayer.

47. The microfluidic device of any one of embodiments 1 to 46, where theinner surface of the base and/or the inner surface of the cover of theenclosure may include glass, silicon, silicon oxide, hafnium oxide,indium tantalum oxide, or aluminum oxide.

48. The microfluidic device of any one of embodiments 1 to 47, where theinner surface of the microfluidic circuit material may includepolydimethylsiloxane (PDMS) or photopatternable silicone (PPS).

49. The microfluidic device of any one of embodiments 1 to 48, wheresubstantially all of the inner surfaces of the enclosure may becovalently modified.

50. The microfluidic device of any one of embodiments 1 to 49, where atleast one inner surface of the base, the cover and the microfluidiccircuit material may have a plurality of third (fourth fifth, etc.)covalently bound surface modifications including a third (fourth, fifth,etc.) linking group, and a third (fourth, fifth, etc.) moiety, where thethird (fourth, fifth, etc.) moiety is a third (fourth, fifth, etc.)surface contact moiety or a third (fourth, fifth, etc.) reactive moiety,where the third (fourth, fifth, etc.) linking group may be differentfrom each of the first and second linking groups and/or the third(fourth, fifth, etc.) moiety may be different from each of the first andsecond moieties.

51. The microfluidic device of any one of embodiments 1 to 50, wherenone of the inner surfaces of the enclosure include gold metal.

52. The microfluidic device of any one of embodiments 1 to 51, where thecover and/or the base may include a semiconductor substrate.

53. The microfluidic device of embodiment 52, where the semiconductorsubstrate may include a dielectrophoresis (DEP) configuration.

54. The microfluidic device of embodiment 53, where the DEPconfiguration may be optically actuated.

55. The microfluidic device of any one of embodiments 52 to 54, wherethe semiconductor substrate may include an electrowetting (EW)configuration.

56. The microfluidic device of embodiment 55, where the fluidic circuitmay include a flow region, fluidically connected to a EW fluidic inletand an EW fluidic outlet, which is configured to contain a flow of an EWfluidic medium.

57. The microfluidic device of embodiment 56, which may further includea chamber including walls enclosing an internal region (which caninclude an isolation region) and an opening to the flow region.

58. The microfluidic device of embodiment 57, where the walls of the atleast one chamber include microfluidic circuit material.

59. The microfluidic device of embodiment 57 or 58, where the walls ofthe at least one chamber may extend from the inner surface of the baseto the inner surface of the cover.

60. The microfluidic device of any one of embodiments 1 to 59, where thecover may be an integral part of the microfluidic circuit material.

61. The microfluidic device of any one of embodiments 1 to 59, where thefirst or the second covalently bound surface modification may have astructure of one of the following formulae: Formula XV; Formula XVI;Formula XVII; Formula XVIII; Formula XIX; Formula XX; Formula XXI;Formula XXII; Formula XXIII; Formula XXIV; Formula XXV; Formula XXVI;Formula XXVII; Formula XXVIII; Formula XXIX; Formula XXXVI; FormulaXXXVII; Formula XXXVIII; Formula XXXIX; and Formula XL.

62. The microfluidic device of embodiment 1, where at least one innersurface of the base, the cover and the microfluidic circuit material ofthe microfluidic device may have a plurality of first covalently boundsurface modifications and a plurality of second covalently boundmodifications of one of the following formulae: Formula XLI; FormulaXLII; Formula XLIII; Formula XLIV; Formula XLIV; Formula XLV; andFormula XLVII.

63. A method of forming a covalently modified surface on at least oneinner surface of a microfluidic device including an enclosure having abase, a cover and microfluidic circuit material defining a fluidiccircuit therein, the method including: contacting the at least one innersurface with a first modifying reagent and a second modifying reagent;reacting the first modifying reagent with a plurality of firstnucleophilic moieties of the at least one inner surface; reacting thesecond modifying reagent with a plurality of second nucleophilicmoieties of the at least one inner surface; and thereby forming at leastone covalently modified surface including first covalently bound surfacemodifications, each including a first linking group and a first moietythat is a first surface contact moiety or a first reactive moiety, andsecond covalently bound surface modifications, each including a secondlinking group and a second moiety that is a second surface contactmoiety or second reactive moiety, where the first linking group isdifferent from the second linking group or the first moiety is differentfrom the second moiety.

64. The method of embodiment 63, where reacting the first modifyingreagent with the at least one inner surface may be performed at the sametime as reacting the second modifying reagent with the at least oneinner surface of the microfluidic device.

65. The method of embodiment 63, where reacting the first modifyingreagent with the at least one inner surface may be performed before orafter reacting the second modifying reagent with the at least one innersurface of the microfluidic device.

66. The method of any one of embodiments 63 to 65, where the firstmodifying reagent may be reacted under conditions allowing the firstmodifying reagent to react with any available nucleophilic moiety of theat least one inner surface, and where the second modifying reagent maybe reacted under conditions allowing the second modifying reagent toreact with any available nucleophilic moiety of the at least one innersurface, such that the first and second covalently bound surfacemodifications are positioned at random upon the at least one innersurface of the microfluidic device.

67. The method of any one of embodiments 63 to 66, where the firstmodifying reagent may be reacted under conditions that promote areaction between the first modifying reagent and nucleophilic moietieslocated on a first region of the at least one surface, and where thesecond modifying reagent may be reacted under conditions that promote areaction between the second modifying reagent and nucleophilic moietieslocated on a second region of the at least one surface, where the firstregion is adjacent to the second region.

68. The method of any one of embodiments 63 to 66, where the firstmodifying reagent is reacted under conditions that promote a reactionbetween the first modifying reagent and nucleophilic moieties locatedwithin any of a plurality of first regions separated from each other onthe at least one surface, and where the second modifying reagent isreacted under conditions that promote a reaction between the secondmodifying reagent and nucleophilic moieties located within a secondregion, where the second region is adjacent to or surrounds each of theplurality of first regions.

69. The method of any one of embodiments 63 to 68, where the fluidiccircuit includes a flow region and a sequestration pen having anisolation region and a connection region, where the connection regionincludes a proximal opening to the flow region and fluidically connectsthe isolation region to the flow region.

70. The method of embodiment 69 where the first modifying reagent may bereacted with first nucleophilic moieties located on a surface of theflow region to form first covalently bound surface modificationsthereon, and where the second modifying reagent may be reacted withsecond nucleophilic moieties located on a surface of the sequestrationpen to form second covalently bound surface modifications thereon.

71. The method of embodiment 70, where the first covalently boundsurface modifications may include a first reactive moiety and the secondcovalently bound surface modifications may include a second reactivemoiety.

72. The method of embodiment 71, where the first and the second reactivemoieties do not react with each other.

73. The method of embodiment 70, where the second covalently boundsurface modifications may include a surface contact moiety which is asupport moiety for adherent cells.

74. The method of embodiment 70 or 73, where the first covalently boundsurface modifications may include a surface contact moiety configured toinhibit or substantially prevent migration of motile cells out of thesequestration pen.

75. The method of any one of embodiments 63 to 74, where forming thecovalently modified surface may include forming a covalently modifiedsurface on substantially all the inner surfaces of the microfluidicdevice.

76. The method of any one of embodiments 63 to 75, where the firstmodifying reagent may have a structure of one of the following formulae:

where: V is —P(O)(OH)₂ or —Si(T)₂W; W is -T, —SH, or —NH₂ and is themoiety configured to form a covalent bond with the at least one innersurface; T is independently OH, OC₁₋₆ alkyl, or halo; R is C₁₋₆ alkyl;L_(fm) is a linker including 1 to 200 non-hydrogen atoms selected fromany combination of silicon, carbon, nitrogen, oxygen, sulfur andphosphorus atoms, and further includes 0 or 1 coupling groups CG; R_(x)is a reactive moiety; n is an integer of 3 to 21, and L_(sm) is a linkerincluding 1 to 200 non-hydrogen atoms selected from any combination ofsilicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms andfurther includes 0, 1, 2, or 3 coupling groups CG.

77. The method of embodiment 76, where W may be OC₁₋₆ alkyl or halo.

78. The method of embodiment 76 or 77, where n may be 7 to 21.

79. The method of any one of embodiments 76 to 78, where T is OC₁₋₃alkyl or halo and/or R is C₁₋₃ alkyl.

80. The method of any one of embodiments 76 to 79, where the reactivemoiety R_(x) may be alkyne, azide, amine, carboxylic acid, biotin, orstreptavidin.

81. The method of any one of embodiments 76 to 79, where the reactivemoiety R_(x) may be alkyne, azide, amine, carboxylic acid, biotin,streptavidin, olefin, trans cyclooctene, s-tetrazine, thiol, maleimide,halide, cyano, isocyanate, epoxide, hydroxyamine, masked hydroxyl, orsulfonyl fluoride.

82. The method of any one of embodiments 76 to 81, where the firstmodifying reagent may have a structure of Formula I, Formula III, orFormula XXXII, and where the surface modifying ligand of the firstmodifying reagent may have a structure of Formula X or Formula XI:

where: CG is a coupling group; L is a linker including a bond or 1 to200 non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms; the sum of L_(sm) and Lis 1 to 200 non-hydrogen atoms, not including atoms of the CG ifpresent; and the surface contact moiety is a moiety configured tosupport cell growth, viability, portability, or any combination thereofin the microfluidic device.

83. The method of embodiment 82, where the surface contact moiety of thefirst modifying reagent may include one or more of an alkyl,fluoroalkyl, monosaccharide, polysaccharide; alcohol, polyalcohol,alkylene ether, polyelectrolytes, amino, carboxylic acid, phosphonicacid, sulfonate anion, carboxybetaines, sulfobetaine, sulfamic acid,amino acid moiety, or cleavable moiety.

84. The method of embodiment 82, where the surface contact moiety of thefirst modifying reagent may include a polyethylene glycol, a dextranmoiety, a proteinaceous moiety, a poly carboxylic acid, or a poly lysinemoiety.

85. The method of any one of embodiments 63 to 84, where the secondmodifying reagent may have a structure of one of the following formulae:

where: V′ is —P(O)(OH)₂ or —Si(T′)₂W′; W′ is -T′, —SH, or —NH₂ and isthe moiety configured to form a covalent bond with the at least oneinner surface; T′ is independently OH, OC₁₋₆ alkyl, or halo; R′ is C₁₋₆alkyl; L′_(fm) is a linker including 1 to 200 non-hydrogen atomsselected from any combination of silicon, carbon, nitrogen, oxygen,sulfur and phosphorus atoms, and further includes 0 or 1 coupling groupsCG; R′_(x) is a reactive moiety; n is an integer of 3 to 21, and L′_(sm)is a linker including 1 to 200 non-hydrogen atoms selected from anycombination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorusatoms and further includes 0, 1, 2, or 3 coupling groups CG.

86. The method of embodiment 85, where W′ is OC₁₋₆ alkyl or halo.

87. The method of embodiment 85 or 86, where n′ is 7 to 21.

88. The method of any one of embodiments 85 to 87, where T′ is OC₁₋₃alkyl or halo and/or R′ is C₁₋₃ alkyl.

89. The method of any one of embodiments 85 to 88, where the reactivemoiety R′_(x) is alkyne, azide, amine, carboxylic acid, biotin, orstreptavidin.

90. The method of any one of embodiments 85 to 88, where the reactivemoiety R′_(x) is alkyne, azide, amine, carboxylic acid, biotin,streptavidin, olefin, trans cyclooctene, s-tetrazine, thiol, maleimide,halide, cyano, isocyanate, epoxide, hydroxyamine, masked hydroxyl, orsulfonyl fluoride.

91. The method of any one of embodiments 85 to 90, where the secondmodifying reagent may have a structure of Formula I′, Formula III′, orFormula XXXII′, and where the surface modifying ligand of the secondmodifying reagent may have a structure of Formula X or Formula XI:

where: CG is a coupling group; L is a linker including a bond or 1 to200 non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms; the sum of L_(sm) and Lis 1 to 200 non-hydrogen atoms, not including atoms of the CG ifpresent; and the surface contact moiety is a moiety configured tosupport cell growth, viability, portability, or any combination thereofin the microfluidic device.

92. The method of embodiment 91, where the surface contact moiety of thesecond modifying reagent may include one or more of an alkyl,fluoroalkyl, monosaccharide, polysaccharide; alcohol, polyalcohol,alkylene ether, polyelectrolytes, amino, carboxylic acid, phosphonicacid, sulfonate anion, carboxybetaines, sulfobetaine, sulfamic acid,amino acid moiety, or cleavable moiety.

93. The method of embodiment 91, where the surface contact moiety of thefirst modifying reagent may include a polyethylene glycol, a dextranmoiety, a proteinaceous moiety, a poly carboxylic acid, or a poly lysinemoiety.

94. The method of embodiment 82 or 91, where the surface contact moietyof the first modifying regent and/or the second modifying reagent maysupport expansion of adherent cells and/or permit export of adherentcells cultured thereupon.

95. The method of embodiment 82 or 91, where the surface contact moietyof the first modifying reagent and/or the second modifying reagent mayinhibit motile cells from entering a selected region within themicrofluidic device.

96. The method of any one of embodiments 76 to 95, where the firstmodifying reagent may have a structure of Formula I, Formula III, orFormula XXXII, and where the second modifying reagent may have astructure of Formula IV′, Formula VI′, or Formula XXXIII′.

97. The method of any one of embodiments 76 to 95, where the firstmodifying reagent may have a structure of Formula IV, Formula VI, orFormula XXXIII, and where the second modifying reagent may have astructure of Formula I′, Formula III′, or Formula XXXII′.

98. The method of any one of embodiments 74 to 95 further includingcontacting the at least one covalently modified surface with a secondaryfunctionalizing reagent of Formula XXXIV

RP-L_(fm)-R_(x2)  Formula XXXIV; and

reacting the secondary functionalizing reagent with reactive moieties onthe first or second covalently bound surface modifications of the atleast one covalently modified surface to form a further modifiedsurface,

where: RP is a reaction pair moiety for reacting with the reactivemoiety of Formula XXXIII, Formula XXXIII′, Formula IV, Formula IV′,Formula VI, or Formula VI′; R_(x2) is a reactive moiety selected to notreact with the reactive moiety of Formula XXXIII, Formula XXXIII′,Formula IV, Formula IV′, Formula VI, or Formula VI′; and L_(fm) is alinker including 1 to 200 non-hydrogen atoms selected from anycombination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorusatoms, and further includes 0 or 1 coupling groups CG.

99. The method of claim 98, wherein contacting the at least onecovalently modified surface with the secondary functionalizing reagentof Formula XXXIV comprises contacting the at least one covalentlymodified surface with a solution comprising the secondaryfunctionalizing reagent.

100. The method of any one of embodiments 76 to 99, further includingcontacting the at least one covalently modified surface with a surfacemodifying reagent, and reacting the surface modifying reagent withreactive moieties on the at least one covalently modified surface or thefurther modified surface.

101. The method of embodiment 100, where the surface modifying reagentmay have a structure of Formula XII:

RP-L-surface contact moiety  Formula XII

where: RP is a reaction pair moiety; the surface contact moiety is amoiety configured to support cell growth, viability, portability, or anycombination; and L is a linker including a bond or 1 to 200 non-hydrogenatoms selected from any combination of silicon, carbon, nitrogen,oxygen, sulfur and phosphorus atoms, and includes 0 or 1 coupling groupsCG.

102. The method of any one of embodiments 63 to 101, where forming theat least one covalently modified surface may be performed after assemblyof the microfluidic device.

103. The method of any one of embodiments 63 to 101, where forming theat least one covalently modified surface may be performed beforeassembly of the microfluidic device.

104. The method of any one of embodiments 63 to 101, further including:forming a first modified surface of one of the base or the cover beforeassembly of the microfluidic device; assembling the microfluidic device,where assembling includes assembling the first covalently modifiedsurface of one of the base or the cover with the microfluidic circuitmaterial and the unmodified one of the cover or base; and forming asecond modified surface on an unmodified surface of the assembledmicrofluidic device.

105. The method of any one of embodiments 63 to 104, where the firstnucleophilic moieties may be hydroxide, amino or thiol, and/or where thesecond nucleophilic moiety is hydroxide, amino or thiol.

106. The method of any one of embodiments 63 to 104, where an innersurface of the base and/or cover may be a metal, metal oxide, glass,polymer, or any combination thereof.

107. The method of any one of embodiments 63 to 106, where themicrofluidic circuit material may be a polymer.

108. The method of embodiment 107, where the microfluidic circuitmaterial may be polydimethoxysilane (PDMS) or photopatternable silicone(PPS).

109. The method of any one of embodiments 63 to 108, where contactingincludes contacting the at least one inner surface with a liquidsolution containing the first modifying reagent and/or the secondmodifying reagent.

110. The method of any one of embodiments 63 to 109, where contactingincludes contacting the at least one inner surface with a vapor phasecontaining the first modifying reagent and/or the second modifyingreagent.

111. The method of embodiment 110, where contacting may includecontacting the at least one inner surface with the first and/or secondmodifying reagent in the vapor phase in the presence of a controlledamount of water vapor.

112. The method of embodiment 111, where magnesium sulfate heptahydratemay provide the controlled amount of water vapor.

113. The method of any one of embodiments 110 to 112, where contactingmay include contacting the at least one inner surface with the firstand/or second modifying reagent in the vapor phase, in an environmentunder reduced pressure relative to atmospheric pressure.

114. The method of any one of embodiments 63 to 113, where each of theat least one inner surface is pre-treated to introduce an oxide moiety.

115. The method of any one of embodiments 76 to 101, where n is 9, 14,or 16.

116. The method of any one of embodiments 76 to 101, where n is 9.

117. The method of any one of embodiments 85 to 101, where n′ equals 9,11, 14, 16, 18, or n+2.

118. The method of any one of embodiments 98 to 101, where reacting theat least one covalently modified surface with a surface modifyingreagent or a secondary functionalizing reagent is performed bycontacting the at least one covalently modified with a solutionincluding the surface modifying reagent or the secondary functionalizingreagent.

119. The method of embodiment 118, where the solution including thesurface modifying reagent or the functionalizing reagent may furtherinclude a Cu(I) salt.

120. The method of embodiment 118, where reacting the at least onecovalently modified surface with the surface modifying reagent or thefunctionalizing reagent may be performed in the absence of copper.

121. The method of any one of embodiments 63 to 120, where forming theat least one covalently modified surface may include forming a monolayerincluding first covalently bound surface modifications and/or secondcovalently bound surface modifications.

122. The method of any one of embodiments 63 to 121, where forming theat least one covalently modified surface may include covalently bindingmore than one kind of proteinaceous moiety to the at least onecovalently modified surface.

123. The method of any one of embodiments 63 to 122, where the cover ofthe microfluidic device may be an integral part of the microfluidiccircuit material.

124. The method of any one of embodiments 63 to 123, where the cover orthe base of the microfluidic device may include a DEP configuration.

125. The method of embodiment 124, where the DEP configuration may beoptically actuated.

126. A method of forming different covalently modified surfaces in aregioselective manner within a microfluidic device, where themicrofluidic device comprises an enclosure having a base, a cover, and amicrofluidic circuit material defining a microfluidic circuit therein,where the microfluidic circuit comprises a flow region and asequestration pen, and where the sequestration pen comprises anisolation region and a connection region, the connection regioncomprising a proximal opening to the flow region and fluidicallyconnecting the isolation region to the flow region, the methodcomprising: flowing a first modifying reagent through the flow regionunder conditions such that the first modifying reagent does not enterthe isolation region of the sequestration pen; reacting the firstmodifying reagent with nucleophilic moieties on at least one surface ofthe flow region, thereby forming a first modified surface within theflow region, where the first modified surface does not extend into theisolation region of the sequestration pen; flowing a second modifyingreagent through the flow region under conditions such that the secondmodifying reagent enters into the isolation region of the sequestrationpen; and reacting the second modifying reagent with nucleophilicmoieties on at least one surface of the isolation region of thesequestration pen, thereby forming a second modified surface within theisolation region of the sequestration pen, where the first modifyingreagent does not have the same structure as the second modifyingreagent.

127. The method of embodiment 126, where the conditions for flowing thefirst modifying reagent through the flow region comprise applying anegative pressure to the flow region.

128. The method of embodiment 127, where flowing the first modifyingreagent comprises flowing a solution that comprises the first modifyingreagent through the flow region at a rate of about 10 mm/sec or higher(e.g., at least 1 mm/sec; at least 5 mm/sec; at least 10 mm/sec; atleast 20 mm/sec; at least 40 mm/sec; at least 50 mm/sec; or any rangedefined by two of the foregoing values, for example, about 1 mm/sec toabout 50 mm/sec, or about 10 mm/sec to about 20 mm/sec).

129. The method of embodiment 126, where the conditions for flowing thefirst modifying reagent through the flow region comprise applying apositive pressure to the flow region.

130. The method of embodiment 129, where flowing the first modifyingreagent comprises flowing a solution that comprises the first modifyingreagent through the flow region at a rate of about 2 mm/sec or less(e.g., less than about 1.5 mm/sec; less than about 1.0 mm/sec; less thanabout 0.5 mm/sec; or any range defined by two of the foregoing values,for example, about 0.5 mm/sec to about 2 mm/sec, or about 1 mm/sec toabout 1.5 mm/sec).

131. The method of embodiment 129 or 130, where flowing the firstmodifying reagent comprises flowing a solution that comprises the firstmodifying reagent through the flow region, and where the solutioncomprises a surfactant (e.g., a non-ionic surfactant, such as a Brijsurfactant (e.g., Brij L4); the surfactant can have ahydrophile-lipophile balance (HLB) of about 8 to about 12 (e.g., about 8to about 10, or about 9).

132. The method of any one of embodiments 126 to 131, where the secondmodifying reagent does not substantially react with moieties on thesurfaces of the flow region.

133. The method of any one of embodiments 126 to 132, where: the firstmodifying reagent comprises a first connecting moiety and a firstmodifying moiety, the first modifying moiety comprising a first surfacecontact moiety or a first reactive moiety; and the second modifyingreagent comprises a second connecting moiety and a second modifyingmoiety, the second modifying moiety comprising a second surface contactmoiety or a second reactive moiety, where the first connecting moiety isdifferent than the second connecting moiety and/or the first modifyingmoiety is different from the second modifying moiety.

134. The method of any one of embodiments 126 to 133, wherein the firstmodifying reagent has a structure of one of the following formulae:

where: V is —P(O)(OH)₂ or —Si(T)₂W; W is -T, —SH, or —NH₂ and is themoiety configured to form a covalent bond with the at least one surfaceof the flow region; T is independently OH, OC₁₋₆ alkyl, or halo; R isC₁₋₆ alkyl; L_(fm) is a linker comprising 1 to 200 non-hydrogen atomsselected from any combination of silicon, carbon, nitrogen, oxygen,sulfur and phosphorus atoms, and further comprises 0 or 1 couplinggroups CG; R_(x) is a reactive moiety; n is an integer of 3 to 21; andL_(sm) is a linker comprising 1 to 200 non-hydrogen atoms selected fromany combination of silicon, carbon, nitrogen, oxygen, sulfur andphosphorus atoms, and further comprises 0, 1, 2, or 3 coupling groupsCG.

135. The method of embodiment 134, where W is OC₁₋₆ alkyl or halo.

136. The method of embodiment 134 or 135, where n is 7 to 21.

137. The method of any one of embodiments 134 to 136, where T is OC₁₋₃alkyl or halo and/or R is C₁₋₃ alkyl.

138. The method of any one of embodiments 134 to 137, where the reactivemoiety R_(x) is alkyne, azide, amine, carboxylic acid, biotin,streptavidin, olefin, trans cyclooctene, s-tetrazine, thiol, maleimide,halide, cyano, isocyanate, epoxide, hydroxyamine, masked hydroxyl, orsulfonyl fluoride.

139. The method of any one of embodiments 134 to 137, where the reactivemoiety R_(x) is alkyne, azide, amine, carboxylic acid, biotin, orstreptavidin.

140. The method of any one of embodiments 134 to 139, where the firstmodifying reagent has a structure of Formula I, Formula III, or FormulaXXXII, and wherein the surface modifying ligand of the first modifyingreagent has a structure of Formula X or Formula XI:

where: CG is a coupling group; L is a linker comprising a bond or 1 to200 non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms; the sum of L_(sm) and Lis 1 to 200 non-hydrogen atoms, not including atoms of the CG ifpresent; and the surface contact moiety is a moiety configured tosupport cell growth, viability, portability, or any combination thereofin the microfluidic device.

141. The method of embodiment 140, where the surface contact moietycomprises one or more of an alkyl, fluoroalkyl, monosaccharide,polysaccharide; alcohol, polyalcohol, alkylene ether, polyelectrolytes,amino, carboxylic acid, phosphonic acid, sulfonate anion,carboxybetaines, sulfobetaine, sulfamic acid, amino acid moiety, orcleavable moiety.

142. The method of embodiment 140, where the surface contact moietycomprises a polyethylene glycol, a dextran moiety, a proteinaceousmoiety, a poly carboxylic acid, or a poly lysine moiety.

143. The method of any one of embodiments 126 to 142, where the secondmodifying reagent has a structure of one of the following formulae:

where: V′ is —P(O)(OH)₂ or —Si(T′)₂W′; W′ is -T′, —SH, or —NH₂ and isthe moiety configured to form a covalent bond with the at least oneinner surface; T′ is independently OH, OC₁₋₆ alkyl, or halo; R′ is C₁₋₆alkyl; L′_(fm) is a linker comprising 1 to 200 non-hydrogen atomsselected from any combination of silicon, carbon, nitrogen, oxygen,sulfur and phosphorus atoms, and further comprises 0 or 1 couplinggroups CG; R′_(x) is a reactive moiety; n is an integer of 3 to 21, andL′_(sm) is a linker comprising 1 to 200 non-hydrogen atoms selected fromany combination of silicon, carbon, nitrogen, oxygen, sulfur andphosphorus atoms and further comprises 0, 1, 2, or 3 coupling groups CG.

144. The method of embodiment 143, where W′ is OC₁₋₆ alkyl or halo.

145. The method of embodiment 143 or 144, where n′ is 7 to 21.

146. The method of any one of embodiments 143 to 145, where T′ is OC₁₋₃alkyl or halo and/or R′ is C₁₋₃ alkyl.

147. The method of any one of embodiments 143 to 146, where the reactivemoiety R′_(x) is alkyne, azide, amine, carboxylic acid, biotin, orstreptavidin.

148. The method of any one of embodiments 143 to 147, where the reactivemoiety R′_(x) is alkyne, azide, amine, carboxylic acid, biotin,streptavidin, olefin, trans cyclooctene, s-tetrazine, thiol, maleimide,halide, cyano, isocyanate, epoxide, hydroxyamine, masked hydroxyl, orsulfonyl fluoride.

149. The method of any one of embodiments 143 to 147, where the secondmodifying reagent has a structure of Formula I′, Formula III′, orFormula XXXII′, and where the surface modifying ligand′ of the secondmodifying reagent has a structure of Formula X or Formula XI:

where: CG is a coupling group; L is a linker comprising a bond or 1 to200 non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms; the sum of L_(sm) and Lis 1 to 200 non-hydrogen atoms, not including atoms of the CG ifpresent; and the surface contact moiety is a moiety configured tosupport cell growth, viability, portability, or any combination thereofin the microfluidic device.

150. The method of embodiment 149, where the surface contact moiety ofthe second modifying reagent comprises one or more of an alkyl,fluoroalkyl, monosaccharide, polysaccharide; alcohol, polyalcohol,alkylene ether, polyelectrolytes, amino, carboxylic acid, phosphonicacid, sulfonate anion, carboxybetaines, sulfobetaine, sulfamic acid,amino acid moiety, or cleavable moiety.

151. The method of embodiment 149, where the surface contact moiety ofthe second modifying reagent comprises a polyethylene glycol, a dextranmoiety, a proteinaceous moiety, a poly carboxylic acid, or a poly lysinemoiety.

152. The method of any one of embodiments 134 to 151, where the surfacecontact moiety of the second modifying reagent supports expansion ofadherent cells and/or permits export of adherent cells culturedthereupon.

153. The method of any one of embodiments 134 to 151, where the surfacecontact moiety of the first modifying reagent inhibits or substantiallyprevents motile cells from entering the flow region of the microfluidicdevice.

154. The method of any one of embodiments 143 to 153, where the firstmodifying reagent has a structure of Formula I, Formula III, or FormulaXXXII, and where the second modifying reagent has a structure of FormulaIV′, Formula VI′, or Formula XXXIII′.

155. The method of any one of embodiments 143 to 153, where the firstmodifying reagent has a structure of Formula IV, Formula VI, or FormulaXXXIII, and where the second modifying reagent has a structure ofFormula I′, Formula III′, or Formula XXXII′.

156. The method of any one of embodiments 126 to 154, where the secondmodified surface within the isolation region of the sequestration pencomprises second covalently bound surface modifications each having astructure of Formula XXX′, Formula V′, or Formula VII′.

157. The method of embodiment 156 further comprising contacting thesecond modified surface with a surface modifying reagent of Formula XII

RP-L-surface contact moiety  Formula XII;

and reacting the second covalently bound surface modifications of thesecond modified surface with the surface modifying reagent to form afurther modified surface within the isolation region of thesequestration pen, where: RP is a reaction pair moiety; the surfacecontact moiety is a moiety configured to support cell growth, viability,portability, or any combination thereof; and L is a linker, wherein Lcomprises a bond or 1 to 200 non-hydrogen atoms selected from anycombination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorusatoms, and further comprises 0 or 1 coupling groups CG.

158. The method of embodiment 157, where contacting the second modifiedsurface with the surface modifying reagent of Formula XII comprises:flowing a solution comprising the surface modifying reagent into theflow region; and allowing the surface modifying reagent to diffuse intothe isolation region of the sequestration pen and contact the secondmodified surface.

159. The method of any one of embodiments 156 to 158, where the firstmodified surface in the flow region comprises first covalently boundsurface modifications each having a structure of Formula XXXI, FormulaVIII, or Formula IX.

160. The method of embodiment 156, further comprising contacting thesecond modified surface with a secondary functionalizing reagent ofFormula XXXIV

RP-L_(fm)-reactive moiety₂  Formula XXXIV;

and reacting the secondary functionalizing reagent with reactivemoieties on the second covalently bound surface modifications of thesecond modified surface to form a further modified surface within theisolation region of the sequestration pen, where: RP is a reaction pairmoiety for reacting with the reactive moiety of Formula XXX, Formula V,or Formula VII; R_(x2) is a reactive moiety selected to not react withthe reactive moiety of the second modified surface; and L_(fm) is alinker comprising 1 to 200 non-hydrogen atoms selected from anycombination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorusatoms and further comprises 0 or 1 coupling groups CG.

161. The method of embodiment 160, where contacting the second modifiedsurface with the secondary functionalizing reagent of Formula XXXIVcomprises: flowing a solution comprising the secondary functionalizingreagent into the flow region; and allowing the secondary functionalizingreagent to diffuse into the isolation region of the sequestration penand contact the second modified surface.

162. The method of embodiment 160 or 161, where the second covalentlybound surface modifications that reacted with the secondaryfunctionalizing reagent each comprise 1 or 2 CG.

163. The method of any one of embodiments 126 to 162, where thenucleophilic moieties on the surface(s) of the flow region are selectedfrom hydroxide, amino, and thiol; and/or where the nucleophilic moietieson the surface(s) of the sequestration pen are selected from hydroxide,amino, and thiol.

164. The method of any one of embodiments 126 to 163, where themicrofluidic circuit comprises a plurality of sequestration pens, eachtreated to form at least one second modified or further modified surfacetherein.

165. The method of any one of embodiments 126 to 164, where an innersurface of the base and/or cover is a metal, metal oxide, glass,polymer, or any combination thereof.

166. The method of any one of embodiments 126 to 165, where themicrofluidic circuit material is a polymer.

167. The method of embodiment 166, where the microfluidic circuitmaterial is polydimethoxysilane (PDMS) or photopatternable silicone(PPS).

168. The method of any one of embodiments 126 to 167, where the cover ofthe microfluidic device is an integral part of the microfluidic circuitmaterial.

169. The method of any one of embodiments 126 to 168, where each of theinner surfaces of the base, cover, and microfluidic circuit material ispre-treated to introduce an oxide moiety.

170. The method of any one of embodiments 134 to 162, where n is 9, 14,or 16.

171. The method of any one of embodiments 134 to 162, where n is 9.

172. The method of any one of embodiments 143 to 162, where n′ equals 9,11, 14, 16, 18, or n+2.

173. The method of embodiment 158 or 161, where the solution comprisingthe surface modifying reagent or the secondary functionalizing reagentfurther comprises a Cu(I) salt.

174. The method of embodiment 158 or 161, where the solution comprisingthe surface modifying reagent or the secondary functionalizing reagentis a copper solution.

175. The method of embodiment 156 or 159, where the first covalentlybound surface modifications form a monolayer on the at least one surfaceof the flow region and/or the second covalently bound surfacemodifications form a monolayer on the at least one surface of theisolation region of the sequestration pen.

176. The method of any one of embodiments 126 to 175, where forming thefirst modified surface and/or forming the second modified surfacecomprises introducing more than one kind of proteinaceous moiety.

177. The method of any one of embodiments 126 to 176, where the cover orthe base of the microfluidic device comprises a DEP configuration.

178. The method of claim 177, where the DEP configuration is opticallyactuated.

179. The method of any one of embodiments 126 to 178, where forming thefirst modified surface comprises forming a covalently modified surfaceon substantially all the inner surfaces of the flow region.

180. The method of any one of embodiments 126 to 179, where forming thesecond modified surface comprises forming a covalently modified surfaceon substantially all the inner surfaces of the isolation region of thesequestration pen.

300. A kit including a microfluidic device of any one of embodiments 1to 62.

301. The kit of embodiment 300, further including a surface modifyingreagent having a structure of Formula XII:

RP-L-surface contact moiety  Formula XII:

wherein RP is a reaction pair moiety; surface contact moiety is a moietyconfigured to support cell growth, viability, portability, or anycombination thereof; L is a linker; wherein L may be a bond or 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms, and may further include 0or 1 coupling groups CG.

302. The kit of embodiment 300 or 301, further including a secondaryfunctionalizing reagent having a structure of Formula XXXIV:

RP-L_(fm)-R_(x2)  Formula XXXIV,

where RP is a reaction pair moiety for reacting with the reactive moietyof Formula XXX, Formula V, or Formula VII; R_(x2) is a reactive moietyselected to not react with the reactive moiety of the functionalizedsurface of Formula XXX, Formula V or Formula VII; and, L_(fm) is alinker comprising 1 to 200 non-hydrogen atoms selected from anycombination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorusatoms and may further include 0 or 1 coupling groups CG.

400. A method of synthesizing a compound of Formula IV:

including the step of: reacting a compound having a structure of FormulaXIII:

where h is 1 to 19 with azide ion, thereby producing the compound ofFormula IV, where n is 3 to 21 and R is H or C₁-C₆ alkyl.

401. The method of embodiment 400, where a counter ion to the azide ionmay be sodium.

402. The method of embodiment 400 or 401, where the reaction may beperformed in acetonitrile or DMF.

403. The method of any one of embodiments 400 to 402, where the reactionis performed at ambient temperature.

404. The method of any one of embodiments 400 to 403, where the reactionis performed under an inert atmosphere.

405. A method of synthesizing a compound having a structure of FormulaXIII

including: reacting a compound having a structure of the followingformula:

with a compound having a structure of the formula HSi(OR)₃, in thepresence of a catalyst or an initiator, thereby producing the compoundof Formula XIII, where h is an integer of 1 to 19 and each instance of Ris independently H or C₁ to C₆ alkyl.

406. The method of embodiment 405, where the catalyst is ahydrosilylation catalyst.

407. The method of embodiment 406, where the catalyst isplatinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex,H₂PtCl₆.6H₂O/iPrOH, or tris(triphenylphosphine)rhodium(I) chloride.

408. The method of any one of embodiments 405 to 407, where the catalystis a platinum (0) catalyst.

409. The method of embodiment 408, where the initiator istrialkylborane.

410. The method of any one of embodiments 405 to 409, where the reactionmay be performed in a solution of toluene.

411. The method of any one of embodiments 405 to 410, where the reactionmay be performed under an inert atmosphere.

412. The method of any one of embodiments 405 to 411, where the reactionmay be performed at a temperature in a range of about 60° C. to about110° C.

413. The method of any one of embodiments 405 to 412, where eachinstance of R is Me or Et.

414. The method of any one of embodiments 405 to 413, where h may be 7,12, or 14.

415. The method of any one of embodiments 405 to 414, where eachinstance of R is Me and h is 7.

416. A compound having a structure of Formula IV:

where n is an integer of 7 to 21, and R is independently H or C₁ to C₆alkyl.

417. The compound of embodiment 416, where R is Me, Et or Pr.

418. The compound of embodiment 416 or 417, where each instance of R maybe Me.

419. The compound of any one of embodiments 416 to 418, where n is 9 to21.

420. The compound of any one of embodiments 416 to 419, where n is 9, 14or 16.

421. The compound of any one of embodiments 416 to 420, where n is 9 andeach instance of R is Me.

422. A compound having a structure of Formula XIII:

where h is an integer of 5 to 19 and R is selected independently fromthe group consisting of H and C₁-C₆ alkyl.

423. The compound of embodiment 422, where n is 9 to 21.

424. The compound of embodiment 422 or 423, where h is 7, 12, or 14.

425. The compound of any one of embodiments 422-424, where h is 14 or16.

426. The compound of any one of embodiments 422-425, where each instanceof R may be Me or Et.

427. A compound having a structure of Formula LI:

wherein R is selected independently from the group consisting of H andC₁-C₆ alkyl.

428. A compound having a structure of the Formula LII:

429. A method of synthesizing a compound of Formula L

comprising the step of:

reacting a compound having a structure of the following formula:

-   -   with a compound having the formula SiH(Y)₃ in the presence of a        catalyst or an initiator, thereby producing the compound of        Formula I, wherein n is an integer of 13 to 25; each instance of        Y is independently halo, OH, or OR; and R is C₁ to C₆ alkyl.

430. The method of embodiment 429, where the catalyst is ahydrosilylation catalyst.

431. The method of embodiment 429, where the catalyst is selected fromplatinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex,H₂PtCl₆.6H₂O/iPrOH, and tris(triphenylphosphine)rhodium(I) chloride.

432. The method of any one of embodiments 429 to 431, where the catalystis a platinum (0) catalyst.

433. The method of embodiment 429, where the initiator istrialkylborane.

434. The method of any one of embodiments 429 to 433, where the step ofreacting is performed in a solution of 1, 3-bis-trifluoromethyl benzene.

435. The method of any one of embodiments 429 to 434, where the step ofreacting is performed under an inert atmosphere.

436. The method of any one of embodiments 429 to 435, where the step ofreacting is performed at a temperature in a range of about 60° C. toabout 110° C.

437. The method of any one of embodiments 429 to 436, where eachinstance of Y is Cl, OMe, or OEt.

438. The method of any one of embodiments 429 to 437, where n is 13, 15,16, or 19.

439. A method of synthesizing a compound having a structure of FormulaLII,

comprising the step of:

-   -   reacting 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 9, 9, 10, 10, 11,        11, 12, 12, 13, 13, 14, 14, 15, 15, 16, 16,        16-nonacosafluorohexadec-1-ene with trimethoxysilane in the        presence of a catalyst or initiator; thereby producing the        molecule of Formula LII (trimethoxy (3, 3, 4, 4, 5, 5, 6, 6, 7,        7, 8, 8, 9, 9, 10, 10, 11, 11, 12, 12, 13, 13, 14, 14, 15, 15,        16, 16, 16-nonacosafluorohexadecyl)-silane).

440. The method of embodiment 439, where the catalyst is ahydrosilylation catalyst.

441. The method of embodiment 439, where the catalyst is selected fromplatinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex,H₂PtCl₆.6H₂O/iPrOH, and tris(triphenylphosphine)rhodium(I) chloride.

442. The method of any one of embodiments 439 to 441, where the catalystis a platinum (0) catalyst.

443. The method of embodiment 439, where the initiator istrialkylborane.

444. The method of any one of embodiments 439 to 443, where the step ofreacting is performed in a solution of 1, 3-bis trifluoromethyl benzene.

445. The method of any one of embodiments 439 to 444, where the step ofreacting is performed under an inert atmosphere.

446. The method of any one of embodiments 439 to 445, where the step ofreacting is performed at a temperature in a range of about 60° C. toabout 110° C.

1. A microfluidic device comprising: an enclosure comprising a base, acover, and microfluidic circuit material defining a fluidic circuittherein, wherein at least one inner surface of the base, the cover andthe microfluidic circuit material has a plurality of first covalentlybound surface modifications, each comprising a first linking group, anda first moiety, wherein the first moiety is a first surface contactmoiety or a first reactive moiety; wherein at least one inner surface ofthe base, the cover and the microfluidic circuit material has aplurality of second covalently bound surface modifications, eachcomprising a second linking group, and a second moiety, wherein thesecond moiety is a second surface contact moiety or second reactivemoiety, and wherein the first linking group and the second linking groupare different from each other and/or the first moiety is different fromthe second moiety.
 2. The microfluidic device of claim 1, wherein thefirst moiety and the second moiety are each covalently bound to thesurface via a linking group LG independently selected from —W—Si(OZ)₂O—and —OP(O)₂O—, wherein W is O, S, or N, and wherein Z is a bond to asilicon atom in an adjacent linking group LG or is a bond to thesurface.
 3. The microfluidic device of claim 1, wherein the firstsurface contact moiety comprises one or more of an alkyl, fluoroalkyl,monosaccharide, polysaccharide, alcohol, polyalcohol, alkylene ether,polyelectrolytes, amino, carboxylic acid, phosphonic acid, sulfonateanion, carboxybetaines, sulfobetaine, sulfamic acid, amino acid moiety,or cleavable moiety; and/or wherein the second surface contact moietycomprises one or more of an alkyl, fluoroalkyl, monosaccharide,polysaccharide, alcohol, polyalcohol, alkylene ether, polyelectrolytes,amino, carboxylic acid, phosphonic acid, sulfonate anion,carboxybetaines, sulfobetaine, sulfamic acid, amino acid moiety, orcleavable moiety.
 4. The microfluidic device of claim 1, wherein thefirst surface contact moiety comprises a polyethylene glycol moiety, adextran moiety, a proteinaceous moiety, a poly carboxylic acid, apolylysine moiety, or any combination thereof; and/or wherein the secondsurface contact moiety comprises a polyethylene glycol moiety, a dextranmoiety, a proteinaceous moiety, a poly carboxylic acid, a polylysinemoiety, or any combination thereof.
 5. The microfluidic device of claim1, wherein the first reactive moiety is an alkyne moiety, an azidemoiety, a carboxylic acid moiety, an amine moiety, an olefinic moiety, atetrazinyl moiety, a trans-cyclooctenyl moiety, a thiol moiety, amaleimide moiety, a biotin moiety, a streptavidin moiety, a halidemoiety, a cyano moiety, isocyanate moiety, an epoxide moiety, ahydroxyamine moiety, or a sulfonyl fluoride moiety; and/or wherein thesecond reactive moiety is an alkyne moiety, an azide moiety, acarboxylic acid moiety, an amine moiety, an olefinic moiety, atetrazinyl moiety, a trans-cyclooctenyl moiety, a thiol moiety, amaleimide moiety, a biotin moiety, a streptavidin moiety, a halidemoiety, a cyano moiety, isocyanate moiety, an epoxide moiety, ahydroxyamine moiety, or a sulfonyl fluoride moiety.
 6. The microfluidicdevice of claim 1, wherein each first covalently bound surfacemodification comprises a linker, wherein the linker comprises 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms; and/or wherein eachsecond covalently bound surface modification comprises a linker, whereinthe linker comprises 1 to 200 non-hydrogen atoms selected from anycombination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorusatoms.
 7. The microfluidic device of claim 6, wherein the linker of thefirst covalently bound surface modifications further comprises one ortwo coupling group CG moieties; and/or wherein the linker of the secondcovalently bound surface modifications further comprises one or twocoupling group CG moieties.
 8. The microfluidic device of claim 1,wherein the first covalently bound surface modifications have astructure selected from Formula XXX, Formula V, Formula VII, FormulaXXXI, Formula VIII, and Formula IX:

wherein: LG is —W—Si(OZ)₂O— or —OP(O)₂O—; L_(fm) is a linker comprising1 to 200 non-hydrogen atoms selected from any combination of silicon,carbon, nitrogen, oxygen, sulfur and phosphorus atoms and furthercomprises 0 or 1 coupling groups CG; R_(x) is a reactive moiety; W is O,S, or N; Z is a bond to an adjacent silicon atom or is a bond to thesurface; n is an integer of 3 to 21; L_(sm) is a linker comprising 1 to200 non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms and further comprises 0,1, 2, or 3 coupling groups CG; and

is the surface.
 9. The microfluidic device of claim 8, wherein LG is—W—Si(OZ)₂O—, and wherein W is O.
 10. The microfluidic device of claim8, wherein n is 7 to
 21. 11. The microfluidic device of claim 8, whereinthe reactive moiety R_(x) is alkyne, azide, amine, carboxylic acid,biotin, or streptavidin.
 12. The microfluidic device of claim 8, whereinthe second covalently bound surface modifications have a structureselected from Formula XXX′, Formula V′, Formula VII′, Formula XXXI′,Formula VIII′, and Formula IX′:

wherein: LG′ is —W′—Si(OZ′)₂O— or —OP(O)₂O—; L′_(fm) is a linkercomprising 1 to 200 non-hydrogen atoms selected from any combination ofsilicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms andfurther comprises 0 or 1 coupling groups CG; R′_(x) is a reactivemoiety; W′ is O, S, or N; Z′ is a bond to an adjacent silicon atom or isa bond to the surface; n′ is an integer of 3 to 21; L′_(sm) is a linkercomprising 1 to 200 non-hydrogen atoms selected from any combination ofsilicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms andfurther comprises 0, 1, 2, or 3 coupling groups CG; and

is the surface.
 13. The microfluidic device of claim 12, wherein LG′ is—W′—Si(OZ′)₂O—, and wherein W′ is O.
 14. The microfluidic device ofclaim 12, wherein n′ is 7 to
 21. 15. The microfluidic device of claim12, wherein the reactive moiety R′_(x) is alkyne, azide, amine,carboxylic acid, biotin, or streptavidin.
 16. The microfluidic device ofclaim 1, wherein the first moiety is different from the second moiety.17-29. (canceled)
 30. The microfluidic device of claim 12, wherein thefirst covalently bound surface modifications have a structure selectedfrom Formula XXX, Formula V, and Formula VII, wherein the secondcovalently bound surface modifications have a structure selected fromFormula XXX′, Formula V′, and Formula VII′, wherein the first covalentlybound surface modifications are different from the second covalentlybound surface modifications, and wherein the reactive moiety of thefirst covalently bound surface modifications does not react with thereactive moiety of the second covalently bound surface modifications.31. The microfluidic device of claim 30, wherein the first covalentlybound surface modifications and the second covalently bound surfacemodifications are on a common inner surface of the base, the cover,and/or the microfluidic circuit material.
 32. The microfluidic device ofclaim 31, wherein the common inner surface comprises a first regioncomprising the first covalently bound surface modifications and a secondregion comprising the second covalently bound surface modifications, andwherein the first region is adjacent to the second region.
 33. Themicrofluidic device of claim 31, wherein the common inner surfacecomprises a plurality of first regions comprising the first covalentlybound surface modifications and a second region comprising the secondcovalently bound surface modifications, wherein the first regions of theplurality are separated from each other by the second region.
 34. Themicrofluidic device of claim 1, wherein the fluidic circuit comprises aflow region and a sequestration pen, wherein the sequestration pencomprises an isolation region and a connection region, wherein theconnection region comprises a proximal opening to the flow region andfluidically connects the isolation region to the flow region.
 35. Themicrofluidic device of claim 34, wherein at least one surface of theflow region is modified with the first covalently bound surfacemodifications, wherein at least one surface of the sequestration pen ismodified with the second covalently bound surface modifications.
 36. Themicrofluidic device of claim 35, wherein the second covalently boundsurface modifications comprises a surface contact moiety configured toanchor adherent cells.
 37. The microfluidic device of claim 35, whereinthe first covalently bound surface modifications comprises a surfacecontact moiety configured to inhibit migration of motile cells out ofthe sequestration pen. 38-39. (canceled)
 40. The microfluidic device ofclaim 34, wherein the fluidic circuit further comprises a plurality ofsequestration pens each having at least one inner surface modified withthe first and/or second covalently bound surface modifications.
 41. Themicrofluidic device of claim 1, wherein the first covalently boundsurface modifications and/or the second covalently bound surfacemodifications form a monolayer.
 42. The microfluidic device of claim 1,wherein the inner surface of the base and/or the inner surface of thecover of the enclosure comprises glass, silicon, silicon oxide, hafniumoxide, indium tantalum oxide, or aluminum oxide.
 43. The microfluidicdevice of claim 1, wherein the inner surface of the microfluidic circuitmaterial comprises polydimethylsiloxane (PDMS) or photopatternablesilicone (PPS).
 44. The microfluidic device of claim 1, whereinsubstantially all of the inner surfaces of the enclosure are covalentlymodified. 45-48. (canceled)
 49. The microfluidic device of claim 1,wherein the first or the second covalently bound surface modificationhas a structure of one of the following formulae:

50-104. (canceled)
 105. A method of forming different covalentlymodified surfaces in a regioselective manner within a microfluidicdevice, wherein the microfluidic device comprises an enclosure having abase, a cover, and a microfluidic circuit material defining amicrofluidic circuit therein, wherein the microfluidic circuit comprisesa flow region and a sequestration pen, and wherein the sequestration pencomprises an isolation region and a connection region, the connectionregion comprising a proximal opening to the flow region and fluidicallyconnecting the isolation region to the flow region, the methodcomprising: flowing a first modifying reagent through the flow regionunder conditions such that the first modifying reagent does not enterthe isolation region of the sequestration pen; reacting the firstmodifying reagent with nucleophilic moieties on at least one surface ofthe flow region, thereby forming a first modified surface within theflow region, wherein the first modified surface does not extend into theisolation region of the sequestration pen; flowing a second modifyingreagent through the flow region under conditions such that the secondmodifying reagent enters into the isolation region of the sequestrationpen; and reacting the second modifying reagent with nucleophilicmoieties on at least one surface of the isolation region of thesequestration pen, thereby forming a second modified surface within theisolation region of the sequestration pen, wherein the first modifyingreagent does not have the same structure as the second modifyingreagent.
 106. The method of claim 105, wherein the conditions forflowing the first modifying reagent through the flow region compriseapplying a negative pressure to the flow region.
 107. The method ofclaim 106, wherein flowing the first modifying reagent comprises flowinga solution that comprises the first modifying reagent through the flowregion at a rate of about 10 mm/sec or higher.
 108. The method of claim105, wherein the conditions for flowing the first modifying reagentthrough the flow region comprise applying a positive pressure to theflow region.
 109. The method of claim 108, wherein flowing the firstmodifying reagent comprises flowing a solution that comprises the firstmodifying reagent through the flow region at a rate of about 2 mm/sec orless.
 110. (canceled)
 111. The method of claim 105, wherein the secondmodifying reagent does not substantially react with moieties on thesurfaces of the flow region. 112-147. (canceled)